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Inhibitory role of miR-203 in the angiogenesis of mice with pathological retinal neovascularization disease through downregulation of SNAI2
Journal Pre-proof Inhibitory role of miR-203 in the angiogenesis of mice with pathological retinal neovascularization disease through downregulation of SNAI2
Li Yu, Shuai Wu, Songtian Che, Yazhen Wu, Ning Han PII:
S0898-6568(20)30047-4
DOI:
https://doi.org/10.1016/j.cellsig.2020.109570
Reference:
CLS 109570
To appear in:
Cellular Signalling
Received date:
12 September 2019
Revised date:
14 February 2020
Accepted date:
14 February 2020
Please cite this article as: L. Yu, S. Wu, S. Che, et al., Inhibitory role of miR-203 in the angiogenesis of mice with pathological retinal neovascularization disease through downregulation of SNAI2, Cellular Signalling(2019), https://doi.org/10.1016/ j.cellsig.2020.109570
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© 2019 Published by Elsevier.
Journal Pre-proof
Inhibitory role of miR-203 in the angiogenesis of mice with pathological
retinal
neovascularization
disease
through
downregulation of SNAI2 Li Yu, Shuai Wu, Songtian Che, Yazhen Wu, Ning Han* [email protected] Department of Ophthalmology, the Second Hospital of Jilin University, Changchun 130041, P.R. China Corresponding author at: Department of Ophthalmology, the Second Hospital of Jilin University,
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*
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No.4026, Yatai Street, Nanguan District, Changchun 130041, P.R. China
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ABSTRACT
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Background: Pathological retinal neovascularization is a disease characterized by abnormal angiogenesis in retina that is a major cause of blindness in humans. Previous reports have
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highlighted the involvement of microRNAs (miRNAs) in retinal angiogenesis. Therefore, we aimed
neovascularization.
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at exploring the mechanism underlying miR-203 regulating the progression of pathological retinal
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Methods: Initially, the mouse model of pathological retinal neovascularization disease was established and the hypoxia-induced human retinal microvascular endothelial cells (HRMECs) were generated. Then, miR-203 and SNAI2 expression in HRMECs and retinal tissues was examined. Subsequently, the effects of miR-203 and SNAI2 on viability, migration, apoptosis and angiogenesis of HRMECs were investigated, with the expression of Bax, Ki-67, MMP-2, MMP-9, VEGF and CD34 measured. Finally, the regulation of miR-203 or SNAI2 on GSK-3β/β-catenin pathway was determined through examining the levels of phosphorylated p-GSK-3β and β-catenin. Results: Poorly expressed miR-203 and highly expressed SNAI2 were found in HRMECs and retinal tissues of pathological retinal neovascularization. Importantly, overexpressed miR-203 or silencing SNAI2 inhibited viability, migration and angiogenesis but promoted apoptosis of 1
Journal Pre-proof HRMECs, evidenced by elevated Bax expression but reduced expression of Ki-67, MMP-2, MMP9, VEGF and CD34. Moreover, overexpression of miR-203 was found to repress the GSK-3β/βcatenin pathway by downregulating SNAI2. Conclusion: Collectively, this study demonstrated that overexpression of miR-203 suppressed the angiogenesis in mice with pathological retinal neovascularization disease via the inactivation of GSK-3β/β-catenin pathway by inhibiting SNAI2, which provided a novel therapeutic insight for pathological retinal neovascularization disease.
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Keywords: microRNA-203; SNAI2; Pathological retinal neovascularization; Angiogenesis; GSK-
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3β/β-catenin pathway
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1. Introduction
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Pathological retinal neovascularization (angiogenesis) is considered as the main cause of blindness in various clinical conditions, such as retinopathy of diabetes and retinopathy of
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prematurity [1, 2]. Angiogenesis is crucial in various physiological processes, including tissue
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repair and wound healing, and is also involved in the pathologic development of several vascular
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diseases, such as vascular eye diseases [3, 4]. Moreover, the abnormal production of proangiogenic growth factors is another driving factor of pathological retinal neovascularization, targe ting which could be a potentially effective treatment option for pathological retinal neovascularization. For example, PEGylated-pigment epithelial-derived factor (PEDF) was identified as a novel therapeutic target for protecting against pathological retinal neovascularization [5]. However, available therapeutic options for pathological retinal angiogenesis are still limited currently [6], suggesting that novel effective treatment is in urgent need. Interestingly, microRNAs (miRNAs) were revealed to exert significant effects on the development of ocular neovascularization [7]. It has been recently found that some miRNAs display angiogenic or antiangiogenic roles in ocular neovascular diseases [8]. For instance, miR-30a-5p was reported to be a crucial regulator in retinal angiogenesis [9]. A recent study demonstrated that miR2
Journal Pre-proof 203 is involved in the angiogenesis of cervical cancer [10]. Furthermore, it was also demonstrated that miR-203 inhibited proliferation and migration but enhanced apoptosis of lung cancer cells [11]. The zinc finger protein SNAI2 has been identified as the target of miR-203 [12], which was reported to be involved in multiple cancers. As a member of the Snail family of zinc finger transcription factors, SNAI2 (also Slug) displays crucial function in the epithelial- mesenchymal transformation (EMT) [13]. As depicted in a former study, SNAIL functioned as the main transcriptional factor in regulating EMT of retinal pigment epithelial cells in human choroidal
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neovascularization [14]. Additionally, SNAIL and Slug were showed to be the target genes of Delta-like, which also exhibited suppressive function in angiogenesis [15]. Moreover, glycogen
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synthase kinase 3 beta (GSK-3β), a serine/threonine protein kinase, is crucial in the tumorigenesis
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of various cancers [16]. GSK3β activity is essential for platelet-derived growth factor-DD (PDGFDD) to suppress angiogenesis [17], and β-catenin pathway is also involved in retina-related diseases
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including retinal angiogenesis [18]. Importantly, a previous study revealed that miR-203 could
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mediate the tumorigenesis, angiogenesis and migration of prostate cancer by regulating SNAI2 via GSK-3β/β-catenin pathway [19]. The aforementioned findings might reveal a network of miR-203,
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SNAI2 and GSK-3β/β-catenin pathway in pathological retinal neovascularization disease. Therefore,
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our study was designed to explore whether miR-203 regulated the development of pathological retinal neovascularization disease and the related mechanism with the involvement of SNAI2 and GSK-3β/β-catenin pathway, to provide a novel target for treating pathological retinal neovascularization disease.
2. Methods and materials 2.1. Ethics statement All animal experiments were conducted in accordance with the principles and procedures of Guide for the Care and Use of Laboratory Animal by the National Institutes of Health. 2.2. Establishment of mouse model of pathological retinal neovascularization disease 3
Journal Pre-proof A total of 40 healthy and clean C57BL/6 mice (Shanghai Laboratory Animal Center of the Chinese Academy of Sciences, Shanghai, China) aged 7 days were enrolled in this study, of which 32 mice were used for model establishment. The mice were placed in closed oxygen box and supplied with 80% mixed oxygen, with gas flow modulated. With the oxyge n concentration monitored using oxygen meter twice per day, the mice were exposed to hyperoxia (75 % ± 2%) at 23°C ± 2°C with lactating mice for 5 days, then transferred to normal environment, and fed for 9 days to induce oxygen- induced retinopathy (OIR) model. Meanwhile, the remaining 8 mice were
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fed in normal environment for 17 days as controls. On the 17th day, all mice were euthanized, with their eyeballs collected and retinal tissues extracted. Then, the retinal tissues were fixed, dehydrated,
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embedded in paraffin, and stained with hematoxylin- eosin (HE). The vascular endothelial nucleus
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that pierced the retinal internal limiting membrane (ILM) was counted [20]. 2.3. Hypoxia-induced human retinal microvascular endothelial cells (HRMECs)
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HRMECs (Shanghai Cell Bank of Chinese Academy of Sciences, Shanghai, China) were
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cultured in Dulbecco's Modified Eagle Medium (DMEM) (PM150510, Procell Life Science & Technology Co., Ltd., Wuhan, China) supplemented with 10% fetal bovine serum (FBS), 100 U/mL
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penicillin and 100 mg/mL streptomycin (15140122, Gibco BRL/Invitrogen, Carlsbad, CA, USA).
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The cells were incubated in hypoxic environment with 3% O 2 and 5% CO 2 and sub-cultured to passage 3 for the following experiments [21]. DMEM medium containing 10% FBS was used for cell culture. 2.4. Cell treatment Cells were grouped by delivering with mimic negative control (NC), miR-203 mimic, short hairpin (sh)-NC, sh-SNAI2, overexpression (oe)-NC and oe-SNAI2 individually or jointly. Then, mice were grouped by injection with the above treated-cells. The in vivo transfection in animals was conducted as follows: 12.5 μg nucleic acid (1 μg/μL) was mixed with 25 μL of 10% glucose solution (w/v) (final volume was 50 μL). Then, 25 μL Entranster TM- invivo transfection reagent (18668-11-1, Engreen Biosystem Co., Ltd., China) was 4
Journal Pre-proof mixed with 25 μL of 10% glucose solution (final volume was 50 μL). Subsequently, the diluted nucleic acid solution was incubated with the diluted transfection reagent for 15 min and injected into the mice via distal 1/3 tail vein (100 μg nucleic acid and 50 μL transfection reagent for each mouse). Cell transfection was performed as follows: 1 × 10 6 cells were treated with 50 μg miR-203 mimic or mimic NC using 100 μL Lipofectamine T M 2000 transfection reagent (11668019, Invitrogen, Carlsbad, California, USA) based on the instructions, or transfected with shRNA against
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SNAI2 using Lipofectamine™ RNA iMAX transfection reagent (13778030, Invitrogen). The cells were then cultured in hypoxic environment with 3% O2 and 5% CO2 for 72 h.
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2.5. Dual-luciferase reporter assay
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The synthetic gene fragments wild-type (Wt) or mutant (Mut) 3'-untranslated region (UTR) of SNAI2 were introduced into pMIR-reporter (Beijing Huayueyang Biotechnology Co., Ltd., Beijing,
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China). Next, the luciferase reporter plasmids Wt or Mut were co-transfected with miR-203 mimic
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and mimic NC into HEK-293T cells (The Cell Resource Center of Shanghai Institutes for Biological Sciences Chinese Academy of Sciences, Shanghai, China) respectively. After 48 h, the
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cells were lysed, and luciferase activity was detected using luciferase detection kit (K801-200,
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BioVision Technologies, Claremont, CA, USA) on Glomax20/20 luminometer fluorescence detector (Promega Corporation, Madison, WI, USA). 2.6. Cell counting kit-8 (CCK-8) assay Cells were seeded in a 96-well plate at a density of 2 × 103 cells/well. At 0 h, 24 h, 48 h, 72 h and 96 h after transfection, cells in each well were incubated with 10 μL CCK -8 solution for 4 h at 37°C. Then, the optical density (OD) value was measured at 450 nm using a microplate reader (BioRad, Hercules, CA, USA). 2.7. Flow cytometry After transfection, cells were collected and centrifuged at 1000 r/min for 5 min with the supernatant discarded. Next, the cells were fixed with 70% precooled ethanol at 4°C. With cell 5
Journal Pre-proof concentration adjusted to 1 × 106 cells/mL, 10 mL cells were stained by Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) (556547, shanghai Shuojia Biotechnology Co., Ltd., Shanghai, China) for 15-30 min at 4°C. Lastly, with the excitation wavelength of 480 nm, FITC was measured at 530 nm and PI was measured at more than 575 nm by using a flow cytometer (XL, Beckman Coulter Life Sciences, Brea, CA, USA). The cell apoptosis rate was expressed as the percentage of apoptotic cells in total cells. 2.8. Transwell assay
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The apical chamber of Transwell chamber (3413, Beijing Unique Biotechnology Co., Ltd., Beijing, China) was pre-coated with Matrigel (40111ES08, Yeasen Company, Shanghai, China)
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diluted in serum- free DMEM (Matrigel: DMEM = 1 : 8) for 4-5 h at 37°C. Next, the cells were
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resuspended with serum- free medium (1 × 106 cells/mL), and seeded into the apical chamber, with 3 duplicated wells set in each group. Subsequently, 500 μL DMEM containing 20% FBS was added
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into the basolateral chamber. After 24 h of incubation with 5% CO 2 at 37°C, the Transwell chamber
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was fixed by 5% glutaraldehyde at 4°C and stained by 0.1% crystal violet for 5 min. Once the cells on surface had been removed, cells were observed under an inverted fluorescence microscope
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(TE2000, Nikon, Tokyo, Japan) and photographed with 5 fields randomly selected (× 400). The
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cells passed through the chamber were counted. 2.9. Angiogenesis in vitro
A 48-well plate was coated by Matrigel (Catalog number: 354234, BD Biosciences, Bedford, MA, USA) (100 μL for each well) for 30 min at 37°C. The HRMECs were suspended in Iscoves modified Dulbecco medium (IMDM; PM150510, Procell Life Science&Technology Co., Ltd.) at a density of 1.5 × 105 cells/mL. Then, 100 μL cell suspension was seeded in the coated 48-well plate and incubated for 24 h at 37°C, with 3 duplicated wells set in each group. Lastly, the Axiovision Rel 4.1 software (Carl Zeiss AG, Mainz and Jena, Germany) was applied to calculate the length of tubules as well as the number of tubule branches. 2.10. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) 6
Journal Pre-proof Total RNA was extracted using Trizol (15596026, Invitrogen), and then reversely transcribed into complementary DNA (cDNA) using the Reverse Transcription Kit (RR047A, Takara Holdings Inc., Kyoto, Japan). With SYBR Premix EX Taq kit (RR420A, Takara Holdings Inc.), RT-qPCR was conducted on the ABI 7500 PCR instrument (ABI Company, Foster City, CA, USA). The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) (Table 1). With 3 duplicated wells set in each group, the Ct value of each well was recorded. With β-actin used as internal reference, the fold changes of gene expression were calculated using the 2-ΔΔCt
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relative quantification method [22]. 2.11. Western blot analysis
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The tissues or cells were lysed with radio immunoprecipitation assay lysis buffer containing
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phenylmethanesulfonyl fluoride to collect total protein. After protein concentration was determined using bicinchoninic acid kit (P0010S-1, Beyotime Biotechnology Co., Shanghai, China), proteins
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were dissolved in 2 × sodium dodecyl sulfate (SDS) loading buffer, separated by SDS-
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polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride membrane (ISEQ00010, Merck Millipore, Billerica, Massachusetts, USA). After being blocked with 5%
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skimmed milk, the membrane was incubated at 4 °C overnight with primary rabbit antibodies
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(Abcam Inc., Cambridge, UK) against Bax (1 : 1000, ab32503), Ki-67 (1 : 1000, ab16667), matrix metalloprotease (MMP)-2 (1 : 1000, ab37150), MMP-9 (1 : 1000, ab38898), vascular endothelial growth factor (VEGF, 1: 1000, ab46154), CD34 (1 : 10000, ab81289), GSK-3β (1 : 5000, ab32391), phosphorylated GSK-3β (1 : 1000, ab75745), and β-catenin (1 : 1000, ab32572). The membrane was re-probed with horseradish peroxidase- labeled secondary antibody for 1 h, developed by the electrochemiluminescence kit (BB-3501, Amersham Inc., Buckinghamshire, UK) and images were obtained using gel imager. Lastly, Bio-Rad image analysis system (Bio-Rad Laboratories) was used for photography and Quantity One v4.6.2 software was used for analysis. The relative expression of gene was regarded as the grey value of corresponding protein bands to that of β-actin protein band. 2.12. In Situ Hybridization 7
Journal Pre-proof The 5-μm paraffin- embedded sections of retina were transferred to positively charged slides. miR-203 was determined by in situ hybridization kit (Biochain Institute, Hayward, CA, USA) and digoxin- labeled custom- made mercury locked nucleic acid miRNA detection probes (Exiqon, Vedbaek, Denmark). A scrambled probe was adopted as a control. 2.13. Immunohistochemistry The retinal tissue sections were dewaxed by xylene, dehydrated b y gradient alcohol, and subjected to antigen retrieval with 0.01 M citrate solution for 20 min. Next, the sections were
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balanced by 0.01 M phosphate buffer saline, treated with 3% hydrogen peroxide for 15 min to eliminate endogenous peroxidase, and blocked by 10% goat serum for 20 min at 37°C. After
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removal of serum, the sections were incubated with primary rabbit antibod ies (Abcam Inc.) against
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VEGF (1 : 100, ab2349), CD34 (1 : 250, ab81289) and SNAI2 (1 : 200, ab27568) at 4°C overnight. The sections were re-warmed for 1 h and incubated with secondary antibody goat anti-rabbit
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immunoglobulin G (ab150117, 1 : 1000, Abcam Inc.) at 37°C for 30 min. Subsequently, the sections were incubated with strept avidin-biotin complex (BOSTER Biological Technology Co.
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Ltd., Wuhan, Hubei, China) at 37°C for 30 min, developed by 3, 3’-diaminobenzidine, and stained
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by hematoxylin for 1 min. Lastly, the sections were decolored by 1% hydrochloric ethanol,
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dehydrated, stained by saturated aluminum carbonate, and cleared by xylene for 15 min. The sections were observed under low power lens by two experienced pathologists, and the microvascular density (MVD) was calculated in accordance with the method of Weidner N [23]. The area with the highest MVD (hotspot) was identified, and the microvessels in 5 fields of vision were calculated under high power lens. The MVD level was considered as the mean value of microvessels. 2.14. HE staining The retinal tissues were fixed, embedded by paraffin, cut into 4- µm sections, dewaxed by xylene, and hydrated by ethanol. Next, sections were stained by hematoxylin for 5 min, differentiated by hydrochloric acid ethanol for 30 s, immersed in running water for 15 min or in 8
Journal Pre-proof warm water at about 50°C for 5 min, and stained by eosin for 2 min. Subsequently, the sections were dehydrated and cleared, followed by mounting with neutral balsam. Lastly, the sections were photographed and observed under an inverted microscope (XSP-8CA, Shanghai Medical Optical Instruments Factory Co., Ltd., Shanghai, China). 2.15. Statistical analysis All data were processed by SPSS 21.0 statistical software (IBM Corp. Armonk, NY, USA). Measurement data were expressed as mean ± standard deviation. All data were tested for normal
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distribution and homogeneity of variance. If data conformed to normal distribution and homogeneity of variance, the paired data between two groups were compared by paired t test, and
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the unpaired data between two groups were compared by unpaired t test. Data among multiple
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groups were compared by one-way analysis of variance (ANOVA) with Tukey’s post hoc test used. The repeated measures ANOVA was applied for analyzing data among multiple groups at different
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time points, and the Bonferroni’s post hoc test was conducted. p < 0.05 was considered as
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3. Results
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statistically significant.
3.1. miR-203 is downregulated and SNAI2 is upregulated in mice and HRMECs with pathological retinal neovascularization disease Initially, HE staining was performed to identify whether the OIR mouse model was successfully established. As shown in Figure 1 A, the number of the vascular endothelial nucleus in neovascularization that pierced the retinal ILM was increased in the OIR mice compared with the normal mice (p < 0.05), suggesting the successful establishment of OIR mouse model. Further, RT-qPCR was conducted to detect miR-203 expression in the retinal tissues of OIR mice and in the hypoxia- induced HRMECs. The results showed a poor expression in miR-203 in the retinal tissues of the OIR mice (Figure 1 B) and in the hypoxia- induced HRMECs (Figure 1 C). Meanwhile, In Situ Hybridization displayed that miR-203 was localized in the cytoplasm and was 9
Journal Pre-proof downregulated in the retinal tissues of OIR mice (Figure 1 D). Moreover, immunohistochemistry results documented that SNAI2 was localized in the nucleus and overexpressed in the retinal tissues of OIR mice (Figure 1 E). These results suggested that miR-203 was poorly expressed, while SNAI2 was highly expressed in mice and HRMECs with pathological retinal neovascularization disease. 3.2. Overexpression of miR-203 suppresses the cell viability, migration and angiogenesis but enhances apoptosis of hypoxia-induced HRMECs
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Based on the aforementioned results, miR-203 was poorly expressed in pathological retinal neovascularization disease. In order to explore whether miR-203 affected the biological features of
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pathological retinal neovascularization disease, HRMECs were induc ed by hypoxia, and miR-203
migration and angiogenesis of HRMECs.
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was overexpressed in hypoxia- induced HRMECs, followed by determination of viability, apoptosis,
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RT-qPCR and western blot analysis suggested that miR-203 was highly expressed in HRMECs
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while SNAI2 was poorly expressed after treatment of miR-203 mimic (Figure 2 A-B). Then, Transwell assay, CCK-8 assay and flow cytometry (Figure 2 C-E) revealed that cell migration and
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treated with miR-203 mimic.
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viability were significantly decreased and cell apoptosis was obviously increased in HRMECs
Angiogenesis assay was employed, which showed that miR-203 mimic treatment led to shortened tubule and reduced tubule branch (Figure 2 F). Furthermore, western blot analysis was applied to examine the expression of cell viability-related factor (Ki-67), apoptosis-related factor (Bax), migration-related factor (MMP-2/MMP-9) and angiogenesis-related factors (VEGF and CD34). The results showed that Bax expression was enhanced while Ki-67, MMP-2/MMP-9, VEGF and CD34 expression was diminished in HRMECs following miR-203 mimic treatment (Figure 2 G). Therefore, a conclusion could be drawn that the overexpression of miR-203 inhibited cell viability, migration and angiogenesis but promoted cell apoptosis of hypoxia- induced HRMECs. 3.3. SNAI2 is a target gene of miR-203 10
Journal Pre-proof The TargetScan database (http://www.targetscan.org/vert_71/) was employed to identify the target relationship between miR-203 and SNAI2, which revealed that miR-203 bound to and targeted SNAI2 (Figure 3 A). Subsequently, this target relationship was verified by dual- luciferase reporter assay. As depicted in Figure 3 B, the luciferase activity of Wt-SNAI2-3'UTR was significantly reduced after co-transfection with miR-203 mimic compared with co-transfection with mimic NC (p < 0.05) while the luciferase activity of Mut-SNAI2-3'UTR exhibited no significant difference between co-transfection with miR-203 mimic and mimic NC (p > 0.05). Subsequently,
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western blot analysis was employed to detect SNAI2 expression, and the results revealed a high level of SNAI2 in OIR mice (Figure 3 C-D). Moreover, SNAI2 expression in HRMECs after
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treatment with miR-203 mimic or miR-203 inhibitor was detected by western blot analysis, which
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revealed that SNAI2 was downregulated by miR-203 mimic but upregulated by miR-203 inhibitor (Figure 3 E). Therefore, miR-203 could target and downregulate SNAI2.
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3.4. Overexpression of miR-203 or downregulation of SNAI2 inhibits viability, migration and
catenin pathway
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angiogenesis but enhances apoptosis of hypoxia-induced HRMECs by inactivating GSK-3β/β-
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It has been reported that miR-203 regulates angiogenesis through the GSK-3β/β-catenin
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pathway by modulating SNAI2 [19]. Based on this finding, we explored whether miR-203 influenced the biological features of hypoxia- induced HRMECs through the GSK-3β/β-catenin pathway by regulating SNAI2. The effects of interactions of miR-203, SNAI2 and GSK-3β/βcatenin pathway on viability, apoptosis, migration, and angiogenesis of HRMECs were determined. RT-qPCR (Figure 4 A) presented that miR-203 mimic significantly increased miR-203 expression and decreased SNAI2 expression (p < 0.05), while sh-SNAI2 had no effect on miR-203 expression (p > 0.05) but led to decreased SNAI2 expression (p < 0.05) in hypoxia- induced HRMECs. Co-treatment of sh-SNAI2 and miR-203 mimic further reduced levels of SNAI2 compared with co-treatment of miR-203 mimic and sh-NC in hypoxia-induced HRMECs.
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Journal Pre-proof Then, Transwell assay, CCK-8 assay and flow cytometry were conducted to examine cell migration, viability and apoptosis of hypoxia- induced HRMECs respectively (Figure 4 B-D). In contrast to treatment of both mimic NC and sh-NC, co-treatment of miR-203 mimic and sh-NC and co-treatment of mimic NC and sh-SNAI2 led to obviously lowered cell migration and viability but significantly higher cell apoptosis (p < 0.05). Moreover, the treatment with both miR-203 mimic and sh-SNAI2 resulted in greatly reduced cell migration and viability and obviously enhanced cell apoptosis in comparison with the co-treatment of miR-203 mimic and sh-NC (p < 0.05).
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Besides, angiogenesis assay (Figure 4 E) revealed that the co-treatment of miR-203 mimic and sh-NC and the co-treatment of mimic NC and sh-SNAI2 resulted in decrease of the length of
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tubules and the number of tubule branches in hypoxia- induced HRMECs compared with the co-
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treatment of mimic NC and sh-NC (p < 0.05), and more significant effect was found after the cotreatment of miR-203 mimic and sh-SNAI2 in contrast to the co-treatment of miR-203 mimic and
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sh-NC (p < 0.05).
Additionally, western blot analysis (Figure 4 F) showed that Bax expression was elevated and
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that of Ki-67, MMP-2, MMP-9, VEGF, CD34, phosphorylated GSK-3β and β-catenin was declined
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in HRMECs following the co-treatment of miR-203 mimic and sh-NC or the co-treatment of mimic
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NC and sh-SNAI2 in comparison with the co-treatment miR-203 mimic and sh-NC (p < 0.05), and more obvious change in expression of Bax, Ki-67, MMP-2/MMP-9, VEGF, CD34, phosphorylated GSK-3β and β-catenin was also induced by the co-treatment of miR-203 mimic and sh-SNAI2 compared with the co-treatment miR-203 mimic and sh-NC (p < 0.05). However, the expression of GSK-3β exhibited no significant difference in hypoxia- induced HRMECs after various treatments (p > 0.05). miR-203 and SNAI2 were overexpressed in the hypoxia-induced HRMECs. RT-qPCR (Supplementary Figure 1 A), CCK-8 (Supplementary Figure 1 B), flow cytometry (Supplementary Figure 1 C), Transwell assay (Supplementary Figure 1 D) and angiogenesis in vitro assay (Supplementary Figure 1 E) revealed that forced expression of SNAI2 could negate the effects of miR-203 mimic on cell viability, apoptosis, migration and angiogenesis. Collectively, 12
Journal Pre-proof overexpression of miR-203 suppressed viability, migration and angiogenesis, but promoted apoptosis of hypoxia- induced HRMECs by inactivating the GSK-3β/β-catenin pathway through downregulation of SNAI2. 3.5. Overexpression of miR-203 or silencing SNAI2 inhibits viability and angiogenesis in the retinal tissues of mice with pathological retinal neovascularization disease by inhibiting the GSK3β/β-catenin pathway To further explore the roles of miR-203 and SNAI2 in angiogenesis, OIR mice model was
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established, in which miR-203 was overexpressed or SNAI2 was silenced and retinal tissues were examined.
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RT-qPCR was firstly conducted, the results of which are shown in Figure 5 A. Treatment of
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miR-203 mimic + sh-NC led to obviously enhanced miR-203 expression but reduced SNAI2 expression in retinal tissues of OIR mice (p < 0.05), while treatment of mimic NC + sh-SNAI2
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exerted no effect on miR-203 expression (p > 0.05) but resulted in lowered SNAI2 expression (p <
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0.05). Moreover, miR-203 expression exhibited no significant difference (p > 0.05) but SNAI2 expression was reduced (p < 0.05) after co-treatment of miR-203 mimic and sh-SNAI2 in contrast
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to the co-treatment of miR-203 mimic and sh-NC.
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Next, HE staining showed that the number of the vascular endothelial nucleus in neovascularization that pierced the retinal ILM was reduced and cell viability within retinal ILM was also reduced after the treatment of miR-203 mimic + sh-NC or mimic NC + sh-SNAI2 relative to the co-treatment of mimic NC and sh-NC, and more remarkable effect was found in retinal tissues of OIR mice after treatment with both miR-203 mimic and sh-SNAI2 in comparison with the co-treatment of miR-203 mimic and sh-NC (Figure 5 B). Furthermore, immunohistochemistry was employed to measure the expression of VEGF and CD34 and the MVD level. Figure 5 C and D exhibited that decreased expression of VEGF and CD34 expression and MVD level were observed in retinal tissues of OIR mice after the treatment of miR-203 mimic and sh-NC or the co-treatment of mimic NC and sh-SNAI2 compared with the 13
Journal Pre-proof co-treatment of mimic NC and sh-NC (all p < 0.05), and more notable change in retinal tissues was induced by co-treatment of miR-203 mimic and sh-SNAI2 in contrast to the treatment with both miR-203 mimic and sh-NC (all p < 0.05). Lastly, western blot analysis was conducted. As illustrated in Figure 5 E, higher expression of Bax but lower expression of Ki-67, MMP-2/MMP-9, VEGF, CD34, phosphorylated p-GSK-3β and β-catenin were observed in retinal tissues of OIR mice after the co-treatment of miR-203 mimic and sh-NC or the co-treatment of mimic NC and sh-SNAI2 than the co-treatment miR-203 mimic and
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sh-NC (p < 0.05), and more significant change in the expression of Ki-67, MMP-2/MMP-9, VEGF, CD34, phosphorylated GSK-3β and β-catenin was induced by the co-treatment of miR-203 mimic
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and sh-SNAI2 compared with the o-treatment miR-203 mimic and sh-NC (p < 0.05). Therefore,
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overexpression of miR-203 or silencing SNAI2 inhibited viability and angiogenesis of the retinal tissues of mice with pathological retinal neovascularization disease through inactivation of the
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4. Discussion
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GSK-3β/β-catenin pathway.
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Retinal neovascularization disease (angiogenesis), one of the main reasons of blindness in the world, occurs due to the damage of retinal blood vessels caused by several conditions [24]. Previously, drugs like the anti-VEGF-A antibody (Ab) (such as bevacizumab and ranibizumab) have been used for the management of retinal angiogenesis [25]; however, despite these advances, the progression is still limited [9]. Besides, although retinal microglia cells have been reported to promote vascular angiogenesis and vasculopathy caused by relative hypoxia, the specific molecular mechanisms underlying retinal angiogenesis is yet to be clarified [26]. Therefore, understanding the pathogenesis of retinal neovascularization is crucial in order to develop highly potent treatments that can manage this condition. Interestingly, miR-203 was clarified to participate in angiogenesis of placentation through suppression of VEGF and VEGFR2 [27]. In addition, GSK -3β has been shown to participate in the mechanism for treating vascular leakage of diabetic retinopathy (DR) in 14
Journal Pre-proof early stage [28]. Thus, we hypothesized that miR-203 participated in the progression including angiogenesis of pathological retinal neovasculariza tion disease. Eventually, the findings from the present study demonstrated that overexpression of miR-203 suppressed the angiogenesis of pathological retinal neovascularization disease via the inactivation of GSK-3β/β-catenin pathway by inhibiting SNAI2. Initially, the results obtained from this study revealed that HRMECs and mice with pathological retinal neovascularization disease presented with downregulated miR-203 and
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upregulated SNAI2. Previous studies have shown decreased expression of miRNAs in retina-related diseases. For instance, miR-384-3p was poorly expressed in retinal tissues of DR mice [29], and
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miR-184 expression was obviously reduced in the retina of OIR mice and thus activated the Wnt
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pathway in ischemia-induced retinal neovascularization [30]. Besides, miR-203 expression was reduced in progenitor cells during retina regeneration [31]. SNAI2 is a zinc finger protein, which
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was highly expressed under the high glucose condition seen in DR, and also led to viability and
al
migration of HRMECs [32]. Furthermore, the target relationship between miR-203 and SNAI2 was verified in this study. Similarly, a previous research revealed that SNAI2 was the target of miR-203
rn
in prostate cells [12]. The target relationship between SNAI2 and miR-203 was also identified in
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prostate cancer cells, and miR-203 and SNAI2 shared association with the mechanism that could function as effective therapy target for prostate cancer [33]. In addition, we found that overexpressing miR-203 or silencing SNAI2 inhibited cell viability, migration and angiogenesis but enhanced cell apoptosis in mice with pathological retinal neovascularization disease through the inactivation of the GSK-3β/β-catenin pathway, evidenced by the lower expression of Ki-67, MMP-2, MMP-9, VEGF, and CD34 but higher expression of Bax. In a previous study, upregulation of miR-203 was reported to suppress the proliferation of progenitor cells in retina regeneration [31]. Moreover, miR-203 was shown to be poorly expressed in lung cancer, and thus overexpressing miR-203 repressed the proliferation, angiogenesis and migration but promoted apoptosis of lung cancer cells [11]. Additionally, SNAI2 was considered as a crucial 15
Journal Pre-proof factor targeted by miR-203 in regulating cell motility, migration and invasion in breast cancer [34]. Overexpression of miR-203 reduced the expression of VEGF and MMP-2 and therefore inhibited the invasion and migration of glioblastoma cells [35]. In previous studies, zinc oxide nanoparticlescaused decrease in MMP-9 expression, suggesting inhibited cell proliferation and migration [36]. miR-203 promoted cell apoptosis in the p53- mutated colon cancer by decreasing Bcl- xL expression and increasing Bax expression [37], which were all consistent with our findings. Furthermore, overexpression of miR-203 and silencing of SNAI2 suppressed angiogenesis in
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mice with pathological retinal neovascularization disease through inactivation of the GSK -3β/βcatenin pathway, which was supported by decreased expression of phosphorylated GSK-3β and β-
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catenin. Previously, miR-203 was reported to downregulate the β-catenin and thus to repress the
e-
activity of osteoblasts in traumatic heterotopic ossification [38]. Also, upregulation of miR-203 greatly reduced the GSK-3α and GSK-3β phosphorylation in liver cancer [39]. Significantly, a prior
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study indicated that overexpressing miR-203 and silencing SNAI2 inhibited proliferation and
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angiogenesis by inactivating the GSK-3β/b-catenin pathway [19]. Therefore, the above data further supports our findings demonstrating the suppressive function of overexpressed miR-203 in
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progression of retinal neovascularization by regulating SNAI2 and GSK-3β/β-catenin pathway.
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In summary, the key findings obtained from the present study signified the crucial effect of miR-203 on the progression of pathological retinal neovascularization disease. This study revealed that miR-203 overexpression inhibited angiogenesis of retinal neovascularization by suppressing GSK-3β/β-catenin pathway through downregulation of SNAI2 (Figure 6). Therefore, this study might provide insights for the development of new and effective treatments for pathological retinal neovascularization disease. Nevertheless, due to the limitations of experimental conditions, we cannot observe the new blood vessels in the OIR model directly through the retinal- flat- mount method in the existing tissues. Therefore, further large-scale studies should be conducted to explore the new blood vessels in the OIR model directly through the retinal-flat- mount method.
16
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Suppleme ntary Figure 1 Overexpressing miR-203 reduces viability, migration and angiogenesis but elevates apoptosis of HRMECs, which was reversed by overexpressing SNAI2. The hypoxiainduced HRMECs were transfected with mimic NC + oe-NC, miR-203 mimic + oe-NC, mimic NC + oe-SNAI2 or miR-203 mimic + oe-SNAI2. A, the transfection efficacy of miR-203 and SNAI2 in HRMECs detected by RT-qPCR. B, the cell viability of HRMECs examined by CCK-8 assay. C, the cell apoptosis of HRMECs detected by flow cytometry. D, the cell migration of HRMECs
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detected by Transwell assay (× 200). E, angiogenesis in HRMECs examined by angiogenesis assay (× 100). * p < 0.05 vs. hypoxia-induced HRMECs treated with mimic NC + oe-NC; # p < 0.05 vs.
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hypoxia-induced HRMECs treated with miR-203 mimic + oe-NC. The measurement data were
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expressed as mean ± standard deviation. The data among multiple groups were analyzed by oneway ANOVA, with Tukey’s post hoc test conducted. The data in Supplementary Figure 1 B at
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different time points were analyzed by repeated measures ANOVA, and the Bonferroni’s post hoc
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Conflicts of Interest
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test was conducted. Each experiment was repeated 3 times.
The authors declare no conflict of interest.
Funding None.
Credit Author Statement Conceptualization: Li Yu, Ning Han; Data curation: Li Yu; Formal analysis: Songtian Che; Investigation: Li Yu; Methodology: Yazhen Wu; Project administration: Ning Han; Resources: Songtian Che; Software: Yazhen Wu; Supervision: Ning Han; Validation: Li Yu, Shuai Wu; 17
Journal Pre-proof Visualization: Shuai Wu; Roles/Writing - original draft: Li Yu, Ning Han; Writing - review & editing: Li Yu, Shuai Wu, Songtian Che, Yazhen Wu, Ning Han
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Figure 1 Poor expression of miR-203 and high expression of SNAI2 are found in mice and HRMECs with pathological retinal neovascularization disease. A, the vascular endothelial nucleus in neovascularization that pierced the retinal ILM measured by HE staining (× 200, the red arrow is the endothelial cell nucleus). B, the miR-203 expression in the retinal tissues of OIR and normal mice detected by RT-qPCR. C, the miR-203 expression in hypoxia- induced HRMECs examined by RT-qPCR. D, the location of miR-203 in retinal tissues evaluated by In Situ Hybridization (× 400). 19
Journal Pre-proof E, the location of SNAI2 in retinal tissues evaluated by immunohistochemistry (× 400). * p < 0.05 vs. the normal mice. The data in figures were measurement data and expressed as mean ± standard deviation. The paired data between two groups conformed to normal distribution and homogeneity of variance were compared by paired t test, and the unpaired data between two groups conformed to normal distribution and homogeneity of variance were compared by unpaired t test. n = 8. Figure 2 Overexpressing miR-203 represses viability, migration and angiogenesis but promotes apoptosis of hypoxia- induced HRMECs. The hypoxia- induced HRMECs were treated with miR-
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203 mimic and mimic NC. A, the miR-203 expression in HRMECs detected by RT-qPCR. C, cell viability of HRMECs determined by CCK-8 assay. D, cell apoptosis of HRMECs measured by flow
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cytometry. E, cell migration of HRMECs detected by Transwell assay (× 200). F, angiogenesis in
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HRMECs measured by angiogenesis assay (× 100). G, the expression of Ki-67, Bax, MMP2/MMP-9, VEGF and CD34 s in HRMECs examined by western blot analysis. * p < 0.05 vs.
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hypoxia-induced HRMECs treated with mimic NC. The values in figures were measurement data
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and expressed as mean ± standard deviation. Data between two groups were compared by unpaired t test, and data in Figure 2C at different time points were analyzed by repeated measure s ANOVA
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with Bonferroni’s post hoc test conducted. Each experiment was repeated 3 times.
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Figure 3 miR-203 targets SNAI2. A, the potential binding site between miR-203 and SNAI2 predicted by TargetScan database. B, the luciferase activity of Wt-SNAI2-3'UTR and Mut-SNAI23'UTR after co-transfection with mimic NC or miR-203 mimic detected by dual- luciferase reporter assay; * p < 0.05 vs. the cells treated with mimic NC. C, the expression of SNAI2 in retina tissues of OIR and normal mice examined by western blot analysis; * p < 0.05 vs. the tissues or cells in OIR and normal mice. E, the expression of SNAI2 in HRMECs after treatment with miR-203 mimic or miR-203 inhibitor; * p < 0.05 vs. the cells treated with inhibitor NC, # p < 0.05 vs. the cells treated with mimic NC. The measurement data were expressed as mean ± standard deviation. Data between two groups were compared by unpaired t test. Each experiment was repeated 3 times.
20
Journal Pre-proof Figure 4 Overexpression of miR-203 or silencing of SNAI2 inactivates the GSK-3β/β-catenin pathway and represses viability, migration and angiogenesis but induces apoptosis of hypoxiainduced HRMECs. Here, the hypoxia- induced HRMECs were transfected with mimic NC + sh-NC, miR-203 mimic + sh-NC, mimic NC + sh-SNAI2 or miR-203 mimic + sh-SNAI2. A, the expression of miR-203 and SNAI2 in HRMECs detected by RT-qPCR. B, the cell viability of HRMECs examined by CCK-8 assay. C, the cell apoptosis of HRMECs detected by flow cytometry. D, the cell migration of HRMECs detected by Transwell assay (× 200). E, angiogenesis in
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HRMECs examined by angiogenesis assay (× 100). F, the expression of Ki-67, Bax, MMP-2/MMP9, VEGF, CD34, phosphorylated GSK-3β and β-catenin in HRMECs measured by western blot
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analysis. * p < 0.05 vs. hypoxia- induced HRMECs treated with mimic NC + sh-NC; # p < 0.05 vs.
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hypoxia-induced HRMECs treated with miR-203 mimic + sh-NC. The measurement data were expressed as mean ± standard deviation. The data among multiple groups were analyzed by one-
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way ANOVA, with Tukey’s post hoc test conducted. The data in Figure 4 B at different time points
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were analyzed by repeated measures ANOVA, and the Bonferroni’s post hoc test was conducted. Each experiment was repeated 3 times.
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Figure 5 Overexpressing miR-203 or silencing SNAI2 suppresses viability and angiogenesis of the
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retinal tissues of mice with pathological retinal neovascularization disease via suppression of the GSK-3β/β-catenin pathway. The OIR mice were treated with mimic NC + sh-NC, miR-203 mimic + sh-NC, mimic NC + sh-SNAI2 or miR-203 mimic + sh-SNAI2. A, the expression of miR-203 and SNAI2 in retinal tissues detected by RT-qPCR. B, the vascular endothelial nucleus in neovascularization that pierced the retinal ILM and the cell viability within retinal ILM observed in retinal tissues by HE staining (× 200, the red arrow is the endothelial cell nucleus). C, the expression of VEGF and CD34 and MVD level in retinal tissues by immunohistochemistry (× 400). D, MVD level in retinal tissues. E, the expression of Ki-67, Bax, MMP-2/MMP-9, VEGF, CD34, phosphorylated GSK-3β and β-catenin in retinal tissues measured by western blot analysis. * p < 0.05 vs. OIR mice treated with mimic NC + sh-NC; # p < 0.05 vs. OIR mice treated with miR-203 21
Journal Pre-proof mimic + sh-NC. The measurement data were expressed as mean ± standard deviation. The data among multiple groups were analyzed by one-way ANOVA, with Tukey’s post hoc test conducted. Each experiment was repeated 3 times. Figure 6 miR-203 regulates the progression of pathological retinal neovascularization disease via the GSK-3β/β-catenin pathway by modulating SNAI2. miR-203 was poorly expressed but SNAI2 was highly expressed in the mouse model of pathological retinal neovascularization disease, and the targeting relationship between miR-203 and SNAI2 was identified. Additionally, overexpressing
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miR-203 suppressed angiogenesis of mice with pathological retinal neovascularization disease through the suppression of the GSK-3β/β-catenin pathway by inhibiting SNAI2.
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Table 1 Primer sequences for RT-qPCR Genes
Sequences
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5’-GCCTCCAAGAAGCCCAACTA-3’
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β-actin
5’-CGAACTGGACACACATACAGTG-3’
R: 5’-CTGAGGATCTCTGGTTGTGGT-3’ F:
SNAI2 (mouse)
5’-ACACTCCAGCTGGCGTGAAATGTTTAGGACCA-3’
R: 5’-CTCAACTGGTGTCGTGGA-3’ F:
SNAI2 (HRMECs)
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R: 5’-GCCGCGTGAAATGTTTAGG-3’ F:
miR-203 (mouse)
5’-GTGCAGGGTCCGAGGT-3’
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F: miR-203 (HRMECs)
R: 5’-GCCGACGATGTCCATACAGT-3’ F:
5'-CTCCATCCTGGCCTCGCTGT-3'
R: 5'-GCTGTCACCTTCACCGTTCC-3'
Notes: RT-qPCR, reverse transcription quantitative polymerase chain reaction; miR-203, microRNA-203; F, forward; R, reverse.
Highlights
miR-203 is poorly expressed in pathological retinal neovascularization disease. SNAI2 is highly expressed in pathological retinal neovascularization disease. miR-203 targets SNAI2 in pathological retinal neovascularization disease. miR-203/SNAI2 axis inhibits angiogenesis via GSK-3β/β-catenin pathway. This study provides novel insights for treating pathological retinal neovascularization.
22
Li Yu, Shuai Wu, Songtian Che, Yazhen Wu, Ning Han PII:
S0898-6568(20)30047-4
DOI:
https://doi.org/10.1016/j.cellsig.2020.109570
Reference:
CLS 109570
To appear in:
Cellular Signalling
Received date:
12 September 2019
Revised date:
14 February 2020
Accepted date:
14 February 2020
Please cite this article as: L. Yu, S. Wu, S. Che, et al., Inhibitory role of miR-203 in the angiogenesis of mice with pathological retinal neovascularization disease through downregulation of SNAI2, Cellular Signalling(2019), https://doi.org/10.1016/ j.cellsig.2020.109570
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© 2019 Published by Elsevier.
Journal Pre-proof
Inhibitory role of miR-203 in the angiogenesis of mice with pathological
retinal
neovascularization
disease
through
downregulation of SNAI2 Li Yu, Shuai Wu, Songtian Che, Yazhen Wu, Ning Han* [email protected] Department of Ophthalmology, the Second Hospital of Jilin University, Changchun 130041, P.R. China Corresponding author at: Department of Ophthalmology, the Second Hospital of Jilin University,
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*
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No.4026, Yatai Street, Nanguan District, Changchun 130041, P.R. China
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ABSTRACT
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Background: Pathological retinal neovascularization is a disease characterized by abnormal angiogenesis in retina that is a major cause of blindness in humans. Previous reports have
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highlighted the involvement of microRNAs (miRNAs) in retinal angiogenesis. Therefore, we aimed
neovascularization.
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at exploring the mechanism underlying miR-203 regulating the progression of pathological retinal
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Methods: Initially, the mouse model of pathological retinal neovascularization disease was established and the hypoxia-induced human retinal microvascular endothelial cells (HRMECs) were generated. Then, miR-203 and SNAI2 expression in HRMECs and retinal tissues was examined. Subsequently, the effects of miR-203 and SNAI2 on viability, migration, apoptosis and angiogenesis of HRMECs were investigated, with the expression of Bax, Ki-67, MMP-2, MMP-9, VEGF and CD34 measured. Finally, the regulation of miR-203 or SNAI2 on GSK-3β/β-catenin pathway was determined through examining the levels of phosphorylated p-GSK-3β and β-catenin. Results: Poorly expressed miR-203 and highly expressed SNAI2 were found in HRMECs and retinal tissues of pathological retinal neovascularization. Importantly, overexpressed miR-203 or silencing SNAI2 inhibited viability, migration and angiogenesis but promoted apoptosis of 1
Journal Pre-proof HRMECs, evidenced by elevated Bax expression but reduced expression of Ki-67, MMP-2, MMP9, VEGF and CD34. Moreover, overexpression of miR-203 was found to repress the GSK-3β/βcatenin pathway by downregulating SNAI2. Conclusion: Collectively, this study demonstrated that overexpression of miR-203 suppressed the angiogenesis in mice with pathological retinal neovascularization disease via the inactivation of GSK-3β/β-catenin pathway by inhibiting SNAI2, which provided a novel therapeutic insight for pathological retinal neovascularization disease.
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Keywords: microRNA-203; SNAI2; Pathological retinal neovascularization; Angiogenesis; GSK-
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3β/β-catenin pathway
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1. Introduction
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Pathological retinal neovascularization (angiogenesis) is considered as the main cause of blindness in various clinical conditions, such as retinopathy of diabetes and retinopathy of
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prematurity [1, 2]. Angiogenesis is crucial in various physiological processes, including tissue
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repair and wound healing, and is also involved in the pathologic development of several vascular
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diseases, such as vascular eye diseases [3, 4]. Moreover, the abnormal production of proangiogenic growth factors is another driving factor of pathological retinal neovascularization, targe ting which could be a potentially effective treatment option for pathological retinal neovascularization. For example, PEGylated-pigment epithelial-derived factor (PEDF) was identified as a novel therapeutic target for protecting against pathological retinal neovascularization [5]. However, available therapeutic options for pathological retinal angiogenesis are still limited currently [6], suggesting that novel effective treatment is in urgent need. Interestingly, microRNAs (miRNAs) were revealed to exert significant effects on the development of ocular neovascularization [7]. It has been recently found that some miRNAs display angiogenic or antiangiogenic roles in ocular neovascular diseases [8]. For instance, miR-30a-5p was reported to be a crucial regulator in retinal angiogenesis [9]. A recent study demonstrated that miR2
Journal Pre-proof 203 is involved in the angiogenesis of cervical cancer [10]. Furthermore, it was also demonstrated that miR-203 inhibited proliferation and migration but enhanced apoptosis of lung cancer cells [11]. The zinc finger protein SNAI2 has been identified as the target of miR-203 [12], which was reported to be involved in multiple cancers. As a member of the Snail family of zinc finger transcription factors, SNAI2 (also Slug) displays crucial function in the epithelial- mesenchymal transformation (EMT) [13]. As depicted in a former study, SNAIL functioned as the main transcriptional factor in regulating EMT of retinal pigment epithelial cells in human choroidal
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neovascularization [14]. Additionally, SNAIL and Slug were showed to be the target genes of Delta-like, which also exhibited suppressive function in angiogenesis [15]. Moreover, glycogen
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synthase kinase 3 beta (GSK-3β), a serine/threonine protein kinase, is crucial in the tumorigenesis
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of various cancers [16]. GSK3β activity is essential for platelet-derived growth factor-DD (PDGFDD) to suppress angiogenesis [17], and β-catenin pathway is also involved in retina-related diseases
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including retinal angiogenesis [18]. Importantly, a previous study revealed that miR-203 could
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mediate the tumorigenesis, angiogenesis and migration of prostate cancer by regulating SNAI2 via GSK-3β/β-catenin pathway [19]. The aforementioned findings might reveal a network of miR-203,
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SNAI2 and GSK-3β/β-catenin pathway in pathological retinal neovascularization disease. Therefore,
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our study was designed to explore whether miR-203 regulated the development of pathological retinal neovascularization disease and the related mechanism with the involvement of SNAI2 and GSK-3β/β-catenin pathway, to provide a novel target for treating pathological retinal neovascularization disease.
2. Methods and materials 2.1. Ethics statement All animal experiments were conducted in accordance with the principles and procedures of Guide for the Care and Use of Laboratory Animal by the National Institutes of Health. 2.2. Establishment of mouse model of pathological retinal neovascularization disease 3
Journal Pre-proof A total of 40 healthy and clean C57BL/6 mice (Shanghai Laboratory Animal Center of the Chinese Academy of Sciences, Shanghai, China) aged 7 days were enrolled in this study, of which 32 mice were used for model establishment. The mice were placed in closed oxygen box and supplied with 80% mixed oxygen, with gas flow modulated. With the oxyge n concentration monitored using oxygen meter twice per day, the mice were exposed to hyperoxia (75 % ± 2%) at 23°C ± 2°C with lactating mice for 5 days, then transferred to normal environment, and fed for 9 days to induce oxygen- induced retinopathy (OIR) model. Meanwhile, the remaining 8 mice were
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fed in normal environment for 17 days as controls. On the 17th day, all mice were euthanized, with their eyeballs collected and retinal tissues extracted. Then, the retinal tissues were fixed, dehydrated,
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embedded in paraffin, and stained with hematoxylin- eosin (HE). The vascular endothelial nucleus
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that pierced the retinal internal limiting membrane (ILM) was counted [20]. 2.3. Hypoxia-induced human retinal microvascular endothelial cells (HRMECs)
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HRMECs (Shanghai Cell Bank of Chinese Academy of Sciences, Shanghai, China) were
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cultured in Dulbecco's Modified Eagle Medium (DMEM) (PM150510, Procell Life Science & Technology Co., Ltd., Wuhan, China) supplemented with 10% fetal bovine serum (FBS), 100 U/mL
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penicillin and 100 mg/mL streptomycin (15140122, Gibco BRL/Invitrogen, Carlsbad, CA, USA).
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The cells were incubated in hypoxic environment with 3% O 2 and 5% CO 2 and sub-cultured to passage 3 for the following experiments [21]. DMEM medium containing 10% FBS was used for cell culture. 2.4. Cell treatment Cells were grouped by delivering with mimic negative control (NC), miR-203 mimic, short hairpin (sh)-NC, sh-SNAI2, overexpression (oe)-NC and oe-SNAI2 individually or jointly. Then, mice were grouped by injection with the above treated-cells. The in vivo transfection in animals was conducted as follows: 12.5 μg nucleic acid (1 μg/μL) was mixed with 25 μL of 10% glucose solution (w/v) (final volume was 50 μL). Then, 25 μL Entranster TM- invivo transfection reagent (18668-11-1, Engreen Biosystem Co., Ltd., China) was 4
Journal Pre-proof mixed with 25 μL of 10% glucose solution (final volume was 50 μL). Subsequently, the diluted nucleic acid solution was incubated with the diluted transfection reagent for 15 min and injected into the mice via distal 1/3 tail vein (100 μg nucleic acid and 50 μL transfection reagent for each mouse). Cell transfection was performed as follows: 1 × 10 6 cells were treated with 50 μg miR-203 mimic or mimic NC using 100 μL Lipofectamine T M 2000 transfection reagent (11668019, Invitrogen, Carlsbad, California, USA) based on the instructions, or transfected with shRNA against
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SNAI2 using Lipofectamine™ RNA iMAX transfection reagent (13778030, Invitrogen). The cells were then cultured in hypoxic environment with 3% O2 and 5% CO2 for 72 h.
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2.5. Dual-luciferase reporter assay
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The synthetic gene fragments wild-type (Wt) or mutant (Mut) 3'-untranslated region (UTR) of SNAI2 were introduced into pMIR-reporter (Beijing Huayueyang Biotechnology Co., Ltd., Beijing,
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China). Next, the luciferase reporter plasmids Wt or Mut were co-transfected with miR-203 mimic
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and mimic NC into HEK-293T cells (The Cell Resource Center of Shanghai Institutes for Biological Sciences Chinese Academy of Sciences, Shanghai, China) respectively. After 48 h, the
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cells were lysed, and luciferase activity was detected using luciferase detection kit (K801-200,
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BioVision Technologies, Claremont, CA, USA) on Glomax20/20 luminometer fluorescence detector (Promega Corporation, Madison, WI, USA). 2.6. Cell counting kit-8 (CCK-8) assay Cells were seeded in a 96-well plate at a density of 2 × 103 cells/well. At 0 h, 24 h, 48 h, 72 h and 96 h after transfection, cells in each well were incubated with 10 μL CCK -8 solution for 4 h at 37°C. Then, the optical density (OD) value was measured at 450 nm using a microplate reader (BioRad, Hercules, CA, USA). 2.7. Flow cytometry After transfection, cells were collected and centrifuged at 1000 r/min for 5 min with the supernatant discarded. Next, the cells were fixed with 70% precooled ethanol at 4°C. With cell 5
Journal Pre-proof concentration adjusted to 1 × 106 cells/mL, 10 mL cells were stained by Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) (556547, shanghai Shuojia Biotechnology Co., Ltd., Shanghai, China) for 15-30 min at 4°C. Lastly, with the excitation wavelength of 480 nm, FITC was measured at 530 nm and PI was measured at more than 575 nm by using a flow cytometer (XL, Beckman Coulter Life Sciences, Brea, CA, USA). The cell apoptosis rate was expressed as the percentage of apoptotic cells in total cells. 2.8. Transwell assay
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The apical chamber of Transwell chamber (3413, Beijing Unique Biotechnology Co., Ltd., Beijing, China) was pre-coated with Matrigel (40111ES08, Yeasen Company, Shanghai, China)
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diluted in serum- free DMEM (Matrigel: DMEM = 1 : 8) for 4-5 h at 37°C. Next, the cells were
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resuspended with serum- free medium (1 × 106 cells/mL), and seeded into the apical chamber, with 3 duplicated wells set in each group. Subsequently, 500 μL DMEM containing 20% FBS was added
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into the basolateral chamber. After 24 h of incubation with 5% CO 2 at 37°C, the Transwell chamber
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was fixed by 5% glutaraldehyde at 4°C and stained by 0.1% crystal violet for 5 min. Once the cells on surface had been removed, cells were observed under an inverted fluorescence microscope
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(TE2000, Nikon, Tokyo, Japan) and photographed with 5 fields randomly selected (× 400). The
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cells passed through the chamber were counted. 2.9. Angiogenesis in vitro
A 48-well plate was coated by Matrigel (Catalog number: 354234, BD Biosciences, Bedford, MA, USA) (100 μL for each well) for 30 min at 37°C. The HRMECs were suspended in Iscoves modified Dulbecco medium (IMDM; PM150510, Procell Life Science&Technology Co., Ltd.) at a density of 1.5 × 105 cells/mL. Then, 100 μL cell suspension was seeded in the coated 48-well plate and incubated for 24 h at 37°C, with 3 duplicated wells set in each group. Lastly, the Axiovision Rel 4.1 software (Carl Zeiss AG, Mainz and Jena, Germany) was applied to calculate the length of tubules as well as the number of tubule branches. 2.10. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) 6
Journal Pre-proof Total RNA was extracted using Trizol (15596026, Invitrogen), and then reversely transcribed into complementary DNA (cDNA) using the Reverse Transcription Kit (RR047A, Takara Holdings Inc., Kyoto, Japan). With SYBR Premix EX Taq kit (RR420A, Takara Holdings Inc.), RT-qPCR was conducted on the ABI 7500 PCR instrument (ABI Company, Foster City, CA, USA). The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) (Table 1). With 3 duplicated wells set in each group, the Ct value of each well was recorded. With β-actin used as internal reference, the fold changes of gene expression were calculated using the 2-ΔΔCt
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relative quantification method [22]. 2.11. Western blot analysis
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The tissues or cells were lysed with radio immunoprecipitation assay lysis buffer containing
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phenylmethanesulfonyl fluoride to collect total protein. After protein concentration was determined using bicinchoninic acid kit (P0010S-1, Beyotime Biotechnology Co., Shanghai, China), proteins
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were dissolved in 2 × sodium dodecyl sulfate (SDS) loading buffer, separated by SDS-
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polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride membrane (ISEQ00010, Merck Millipore, Billerica, Massachusetts, USA). After being blocked with 5%
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skimmed milk, the membrane was incubated at 4 °C overnight with primary rabbit antibodies
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(Abcam Inc., Cambridge, UK) against Bax (1 : 1000, ab32503), Ki-67 (1 : 1000, ab16667), matrix metalloprotease (MMP)-2 (1 : 1000, ab37150), MMP-9 (1 : 1000, ab38898), vascular endothelial growth factor (VEGF, 1: 1000, ab46154), CD34 (1 : 10000, ab81289), GSK-3β (1 : 5000, ab32391), phosphorylated GSK-3β (1 : 1000, ab75745), and β-catenin (1 : 1000, ab32572). The membrane was re-probed with horseradish peroxidase- labeled secondary antibody for 1 h, developed by the electrochemiluminescence kit (BB-3501, Amersham Inc., Buckinghamshire, UK) and images were obtained using gel imager. Lastly, Bio-Rad image analysis system (Bio-Rad Laboratories) was used for photography and Quantity One v4.6.2 software was used for analysis. The relative expression of gene was regarded as the grey value of corresponding protein bands to that of β-actin protein band. 2.12. In Situ Hybridization 7
Journal Pre-proof The 5-μm paraffin- embedded sections of retina were transferred to positively charged slides. miR-203 was determined by in situ hybridization kit (Biochain Institute, Hayward, CA, USA) and digoxin- labeled custom- made mercury locked nucleic acid miRNA detection probes (Exiqon, Vedbaek, Denmark). A scrambled probe was adopted as a control. 2.13. Immunohistochemistry The retinal tissue sections were dewaxed by xylene, dehydrated b y gradient alcohol, and subjected to antigen retrieval with 0.01 M citrate solution for 20 min. Next, the sections were
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balanced by 0.01 M phosphate buffer saline, treated with 3% hydrogen peroxide for 15 min to eliminate endogenous peroxidase, and blocked by 10% goat serum for 20 min at 37°C. After
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removal of serum, the sections were incubated with primary rabbit antibod ies (Abcam Inc.) against
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VEGF (1 : 100, ab2349), CD34 (1 : 250, ab81289) and SNAI2 (1 : 200, ab27568) at 4°C overnight. The sections were re-warmed for 1 h and incubated with secondary antibody goat anti-rabbit
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immunoglobulin G (ab150117, 1 : 1000, Abcam Inc.) at 37°C for 30 min. Subsequently, the sections were incubated with strept avidin-biotin complex (BOSTER Biological Technology Co.
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Ltd., Wuhan, Hubei, China) at 37°C for 30 min, developed by 3, 3’-diaminobenzidine, and stained
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by hematoxylin for 1 min. Lastly, the sections were decolored by 1% hydrochloric ethanol,
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dehydrated, stained by saturated aluminum carbonate, and cleared by xylene for 15 min. The sections were observed under low power lens by two experienced pathologists, and the microvascular density (MVD) was calculated in accordance with the method of Weidner N [23]. The area with the highest MVD (hotspot) was identified, and the microvessels in 5 fields of vision were calculated under high power lens. The MVD level was considered as the mean value of microvessels. 2.14. HE staining The retinal tissues were fixed, embedded by paraffin, cut into 4- µm sections, dewaxed by xylene, and hydrated by ethanol. Next, sections were stained by hematoxylin for 5 min, differentiated by hydrochloric acid ethanol for 30 s, immersed in running water for 15 min or in 8
Journal Pre-proof warm water at about 50°C for 5 min, and stained by eosin for 2 min. Subsequently, the sections were dehydrated and cleared, followed by mounting with neutral balsam. Lastly, the sections were photographed and observed under an inverted microscope (XSP-8CA, Shanghai Medical Optical Instruments Factory Co., Ltd., Shanghai, China). 2.15. Statistical analysis All data were processed by SPSS 21.0 statistical software (IBM Corp. Armonk, NY, USA). Measurement data were expressed as mean ± standard deviation. All data were tested for normal
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distribution and homogeneity of variance. If data conformed to normal distribution and homogeneity of variance, the paired data between two groups were compared by paired t test, and
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the unpaired data between two groups were compared by unpaired t test. Data among multiple
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groups were compared by one-way analysis of variance (ANOVA) with Tukey’s post hoc test used. The repeated measures ANOVA was applied for analyzing data among multiple groups at different
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time points, and the Bonferroni’s post hoc test was conducted. p < 0.05 was considered as
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3. Results
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statistically significant.
3.1. miR-203 is downregulated and SNAI2 is upregulated in mice and HRMECs with pathological retinal neovascularization disease Initially, HE staining was performed to identify whether the OIR mouse model was successfully established. As shown in Figure 1 A, the number of the vascular endothelial nucleus in neovascularization that pierced the retinal ILM was increased in the OIR mice compared with the normal mice (p < 0.05), suggesting the successful establishment of OIR mouse model. Further, RT-qPCR was conducted to detect miR-203 expression in the retinal tissues of OIR mice and in the hypoxia- induced HRMECs. The results showed a poor expression in miR-203 in the retinal tissues of the OIR mice (Figure 1 B) and in the hypoxia- induced HRMECs (Figure 1 C). Meanwhile, In Situ Hybridization displayed that miR-203 was localized in the cytoplasm and was 9
Journal Pre-proof downregulated in the retinal tissues of OIR mice (Figure 1 D). Moreover, immunohistochemistry results documented that SNAI2 was localized in the nucleus and overexpressed in the retinal tissues of OIR mice (Figure 1 E). These results suggested that miR-203 was poorly expressed, while SNAI2 was highly expressed in mice and HRMECs with pathological retinal neovascularization disease. 3.2. Overexpression of miR-203 suppresses the cell viability, migration and angiogenesis but enhances apoptosis of hypoxia-induced HRMECs
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Based on the aforementioned results, miR-203 was poorly expressed in pathological retinal neovascularization disease. In order to explore whether miR-203 affected the biological features of
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pathological retinal neovascularization disease, HRMECs were induc ed by hypoxia, and miR-203
migration and angiogenesis of HRMECs.
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was overexpressed in hypoxia- induced HRMECs, followed by determination of viability, apoptosis,
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RT-qPCR and western blot analysis suggested that miR-203 was highly expressed in HRMECs
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while SNAI2 was poorly expressed after treatment of miR-203 mimic (Figure 2 A-B). Then, Transwell assay, CCK-8 assay and flow cytometry (Figure 2 C-E) revealed that cell migration and
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treated with miR-203 mimic.
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viability were significantly decreased and cell apoptosis was obviously increased in HRMECs
Angiogenesis assay was employed, which showed that miR-203 mimic treatment led to shortened tubule and reduced tubule branch (Figure 2 F). Furthermore, western blot analysis was applied to examine the expression of cell viability-related factor (Ki-67), apoptosis-related factor (Bax), migration-related factor (MMP-2/MMP-9) and angiogenesis-related factors (VEGF and CD34). The results showed that Bax expression was enhanced while Ki-67, MMP-2/MMP-9, VEGF and CD34 expression was diminished in HRMECs following miR-203 mimic treatment (Figure 2 G). Therefore, a conclusion could be drawn that the overexpression of miR-203 inhibited cell viability, migration and angiogenesis but promoted cell apoptosis of hypoxia- induced HRMECs. 3.3. SNAI2 is a target gene of miR-203 10
Journal Pre-proof The TargetScan database (http://www.targetscan.org/vert_71/) was employed to identify the target relationship between miR-203 and SNAI2, which revealed that miR-203 bound to and targeted SNAI2 (Figure 3 A). Subsequently, this target relationship was verified by dual- luciferase reporter assay. As depicted in Figure 3 B, the luciferase activity of Wt-SNAI2-3'UTR was significantly reduced after co-transfection with miR-203 mimic compared with co-transfection with mimic NC (p < 0.05) while the luciferase activity of Mut-SNAI2-3'UTR exhibited no significant difference between co-transfection with miR-203 mimic and mimic NC (p > 0.05). Subsequently,
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western blot analysis was employed to detect SNAI2 expression, and the results revealed a high level of SNAI2 in OIR mice (Figure 3 C-D). Moreover, SNAI2 expression in HRMECs after
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treatment with miR-203 mimic or miR-203 inhibitor was detected by western blot analysis, which
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revealed that SNAI2 was downregulated by miR-203 mimic but upregulated by miR-203 inhibitor (Figure 3 E). Therefore, miR-203 could target and downregulate SNAI2.
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3.4. Overexpression of miR-203 or downregulation of SNAI2 inhibits viability, migration and
catenin pathway
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angiogenesis but enhances apoptosis of hypoxia-induced HRMECs by inactivating GSK-3β/β-
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It has been reported that miR-203 regulates angiogenesis through the GSK-3β/β-catenin
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pathway by modulating SNAI2 [19]. Based on this finding, we explored whether miR-203 influenced the biological features of hypoxia- induced HRMECs through the GSK-3β/β-catenin pathway by regulating SNAI2. The effects of interactions of miR-203, SNAI2 and GSK-3β/βcatenin pathway on viability, apoptosis, migration, and angiogenesis of HRMECs were determined. RT-qPCR (Figure 4 A) presented that miR-203 mimic significantly increased miR-203 expression and decreased SNAI2 expression (p < 0.05), while sh-SNAI2 had no effect on miR-203 expression (p > 0.05) but led to decreased SNAI2 expression (p < 0.05) in hypoxia- induced HRMECs. Co-treatment of sh-SNAI2 and miR-203 mimic further reduced levels of SNAI2 compared with co-treatment of miR-203 mimic and sh-NC in hypoxia-induced HRMECs.
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Journal Pre-proof Then, Transwell assay, CCK-8 assay and flow cytometry were conducted to examine cell migration, viability and apoptosis of hypoxia- induced HRMECs respectively (Figure 4 B-D). In contrast to treatment of both mimic NC and sh-NC, co-treatment of miR-203 mimic and sh-NC and co-treatment of mimic NC and sh-SNAI2 led to obviously lowered cell migration and viability but significantly higher cell apoptosis (p < 0.05). Moreover, the treatment with both miR-203 mimic and sh-SNAI2 resulted in greatly reduced cell migration and viability and obviously enhanced cell apoptosis in comparison with the co-treatment of miR-203 mimic and sh-NC (p < 0.05).
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Besides, angiogenesis assay (Figure 4 E) revealed that the co-treatment of miR-203 mimic and sh-NC and the co-treatment of mimic NC and sh-SNAI2 resulted in decrease of the length of
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tubules and the number of tubule branches in hypoxia- induced HRMECs compared with the co-
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treatment of mimic NC and sh-NC (p < 0.05), and more significant effect was found after the cotreatment of miR-203 mimic and sh-SNAI2 in contrast to the co-treatment of miR-203 mimic and
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sh-NC (p < 0.05).
Additionally, western blot analysis (Figure 4 F) showed that Bax expression was elevated and
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that of Ki-67, MMP-2, MMP-9, VEGF, CD34, phosphorylated GSK-3β and β-catenin was declined
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in HRMECs following the co-treatment of miR-203 mimic and sh-NC or the co-treatment of mimic
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NC and sh-SNAI2 in comparison with the co-treatment miR-203 mimic and sh-NC (p < 0.05), and more obvious change in expression of Bax, Ki-67, MMP-2/MMP-9, VEGF, CD34, phosphorylated GSK-3β and β-catenin was also induced by the co-treatment of miR-203 mimic and sh-SNAI2 compared with the co-treatment miR-203 mimic and sh-NC (p < 0.05). However, the expression of GSK-3β exhibited no significant difference in hypoxia- induced HRMECs after various treatments (p > 0.05). miR-203 and SNAI2 were overexpressed in the hypoxia-induced HRMECs. RT-qPCR (Supplementary Figure 1 A), CCK-8 (Supplementary Figure 1 B), flow cytometry (Supplementary Figure 1 C), Transwell assay (Supplementary Figure 1 D) and angiogenesis in vitro assay (Supplementary Figure 1 E) revealed that forced expression of SNAI2 could negate the effects of miR-203 mimic on cell viability, apoptosis, migration and angiogenesis. Collectively, 12
Journal Pre-proof overexpression of miR-203 suppressed viability, migration and angiogenesis, but promoted apoptosis of hypoxia- induced HRMECs by inactivating the GSK-3β/β-catenin pathway through downregulation of SNAI2. 3.5. Overexpression of miR-203 or silencing SNAI2 inhibits viability and angiogenesis in the retinal tissues of mice with pathological retinal neovascularization disease by inhibiting the GSK3β/β-catenin pathway To further explore the roles of miR-203 and SNAI2 in angiogenesis, OIR mice model was
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established, in which miR-203 was overexpressed or SNAI2 was silenced and retinal tissues were examined.
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RT-qPCR was firstly conducted, the results of which are shown in Figure 5 A. Treatment of
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miR-203 mimic + sh-NC led to obviously enhanced miR-203 expression but reduced SNAI2 expression in retinal tissues of OIR mice (p < 0.05), while treatment of mimic NC + sh-SNAI2
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exerted no effect on miR-203 expression (p > 0.05) but resulted in lowered SNAI2 expression (p <
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0.05). Moreover, miR-203 expression exhibited no significant difference (p > 0.05) but SNAI2 expression was reduced (p < 0.05) after co-treatment of miR-203 mimic and sh-SNAI2 in contrast
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to the co-treatment of miR-203 mimic and sh-NC.
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Next, HE staining showed that the number of the vascular endothelial nucleus in neovascularization that pierced the retinal ILM was reduced and cell viability within retinal ILM was also reduced after the treatment of miR-203 mimic + sh-NC or mimic NC + sh-SNAI2 relative to the co-treatment of mimic NC and sh-NC, and more remarkable effect was found in retinal tissues of OIR mice after treatment with both miR-203 mimic and sh-SNAI2 in comparison with the co-treatment of miR-203 mimic and sh-NC (Figure 5 B). Furthermore, immunohistochemistry was employed to measure the expression of VEGF and CD34 and the MVD level. Figure 5 C and D exhibited that decreased expression of VEGF and CD34 expression and MVD level were observed in retinal tissues of OIR mice after the treatment of miR-203 mimic and sh-NC or the co-treatment of mimic NC and sh-SNAI2 compared with the 13
Journal Pre-proof co-treatment of mimic NC and sh-NC (all p < 0.05), and more notable change in retinal tissues was induced by co-treatment of miR-203 mimic and sh-SNAI2 in contrast to the treatment with both miR-203 mimic and sh-NC (all p < 0.05). Lastly, western blot analysis was conducted. As illustrated in Figure 5 E, higher expression of Bax but lower expression of Ki-67, MMP-2/MMP-9, VEGF, CD34, phosphorylated p-GSK-3β and β-catenin were observed in retinal tissues of OIR mice after the co-treatment of miR-203 mimic and sh-NC or the co-treatment of mimic NC and sh-SNAI2 than the co-treatment miR-203 mimic and
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sh-NC (p < 0.05), and more significant change in the expression of Ki-67, MMP-2/MMP-9, VEGF, CD34, phosphorylated GSK-3β and β-catenin was induced by the co-treatment of miR-203 mimic
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and sh-SNAI2 compared with the o-treatment miR-203 mimic and sh-NC (p < 0.05). Therefore,
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overexpression of miR-203 or silencing SNAI2 inhibited viability and angiogenesis of the retinal tissues of mice with pathological retinal neovascularization disease through inactivation of the
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4. Discussion
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GSK-3β/β-catenin pathway.
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Retinal neovascularization disease (angiogenesis), one of the main reasons of blindness in the world, occurs due to the damage of retinal blood vessels caused by several conditions [24]. Previously, drugs like the anti-VEGF-A antibody (Ab) (such as bevacizumab and ranibizumab) have been used for the management of retinal angiogenesis [25]; however, despite these advances, the progression is still limited [9]. Besides, although retinal microglia cells have been reported to promote vascular angiogenesis and vasculopathy caused by relative hypoxia, the specific molecular mechanisms underlying retinal angiogenesis is yet to be clarified [26]. Therefore, understanding the pathogenesis of retinal neovascularization is crucial in order to develop highly potent treatments that can manage this condition. Interestingly, miR-203 was clarified to participate in angiogenesis of placentation through suppression of VEGF and VEGFR2 [27]. In addition, GSK -3β has been shown to participate in the mechanism for treating vascular leakage of diabetic retinopathy (DR) in 14
Journal Pre-proof early stage [28]. Thus, we hypothesized that miR-203 participated in the progression including angiogenesis of pathological retinal neovasculariza tion disease. Eventually, the findings from the present study demonstrated that overexpression of miR-203 suppressed the angiogenesis of pathological retinal neovascularization disease via the inactivation of GSK-3β/β-catenin pathway by inhibiting SNAI2. Initially, the results obtained from this study revealed that HRMECs and mice with pathological retinal neovascularization disease presented with downregulated miR-203 and
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upregulated SNAI2. Previous studies have shown decreased expression of miRNAs in retina-related diseases. For instance, miR-384-3p was poorly expressed in retinal tissues of DR mice [29], and
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miR-184 expression was obviously reduced in the retina of OIR mice and thus activated the Wnt
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pathway in ischemia-induced retinal neovascularization [30]. Besides, miR-203 expression was reduced in progenitor cells during retina regeneration [31]. SNAI2 is a zinc finger protein, which
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was highly expressed under the high glucose condition seen in DR, and also led to viability and
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migration of HRMECs [32]. Furthermore, the target relationship between miR-203 and SNAI2 was verified in this study. Similarly, a previous research revealed that SNAI2 was the target of miR-203
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in prostate cells [12]. The target relationship between SNAI2 and miR-203 was also identified in
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prostate cancer cells, and miR-203 and SNAI2 shared association with the mechanism that could function as effective therapy target for prostate cancer [33]. In addition, we found that overexpressing miR-203 or silencing SNAI2 inhibited cell viability, migration and angiogenesis but enhanced cell apoptosis in mice with pathological retinal neovascularization disease through the inactivation of the GSK-3β/β-catenin pathway, evidenced by the lower expression of Ki-67, MMP-2, MMP-9, VEGF, and CD34 but higher expression of Bax. In a previous study, upregulation of miR-203 was reported to suppress the proliferation of progenitor cells in retina regeneration [31]. Moreover, miR-203 was shown to be poorly expressed in lung cancer, and thus overexpressing miR-203 repressed the proliferation, angiogenesis and migration but promoted apoptosis of lung cancer cells [11]. Additionally, SNAI2 was considered as a crucial 15
Journal Pre-proof factor targeted by miR-203 in regulating cell motility, migration and invasion in breast cancer [34]. Overexpression of miR-203 reduced the expression of VEGF and MMP-2 and therefore inhibited the invasion and migration of glioblastoma cells [35]. In previous studies, zinc oxide nanoparticlescaused decrease in MMP-9 expression, suggesting inhibited cell proliferation and migration [36]. miR-203 promoted cell apoptosis in the p53- mutated colon cancer by decreasing Bcl- xL expression and increasing Bax expression [37], which were all consistent with our findings. Furthermore, overexpression of miR-203 and silencing of SNAI2 suppressed angiogenesis in
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mice with pathological retinal neovascularization disease through inactivation of the GSK -3β/βcatenin pathway, which was supported by decreased expression of phosphorylated GSK-3β and β-
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catenin. Previously, miR-203 was reported to downregulate the β-catenin and thus to repress the
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activity of osteoblasts in traumatic heterotopic ossification [38]. Also, upregulation of miR-203 greatly reduced the GSK-3α and GSK-3β phosphorylation in liver cancer [39]. Significantly, a prior
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study indicated that overexpressing miR-203 and silencing SNAI2 inhibited proliferation and
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angiogenesis by inactivating the GSK-3β/b-catenin pathway [19]. Therefore, the above data further supports our findings demonstrating the suppressive function of overexpressed miR-203 in
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progression of retinal neovascularization by regulating SNAI2 and GSK-3β/β-catenin pathway.
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In summary, the key findings obtained from the present study signified the crucial effect of miR-203 on the progression of pathological retinal neovascularization disease. This study revealed that miR-203 overexpression inhibited angiogenesis of retinal neovascularization by suppressing GSK-3β/β-catenin pathway through downregulation of SNAI2 (Figure 6). Therefore, this study might provide insights for the development of new and effective treatments for pathological retinal neovascularization disease. Nevertheless, due to the limitations of experimental conditions, we cannot observe the new blood vessels in the OIR model directly through the retinal- flat- mount method in the existing tissues. Therefore, further large-scale studies should be conducted to explore the new blood vessels in the OIR model directly through the retinal-flat- mount method.
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Suppleme ntary Figure 1 Overexpressing miR-203 reduces viability, migration and angiogenesis but elevates apoptosis of HRMECs, which was reversed by overexpressing SNAI2. The hypoxiainduced HRMECs were transfected with mimic NC + oe-NC, miR-203 mimic + oe-NC, mimic NC + oe-SNAI2 or miR-203 mimic + oe-SNAI2. A, the transfection efficacy of miR-203 and SNAI2 in HRMECs detected by RT-qPCR. B, the cell viability of HRMECs examined by CCK-8 assay. C, the cell apoptosis of HRMECs detected by flow cytometry. D, the cell migration of HRMECs
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detected by Transwell assay (× 200). E, angiogenesis in HRMECs examined by angiogenesis assay (× 100). * p < 0.05 vs. hypoxia-induced HRMECs treated with mimic NC + oe-NC; # p < 0.05 vs.
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hypoxia-induced HRMECs treated with miR-203 mimic + oe-NC. The measurement data were
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expressed as mean ± standard deviation. The data among multiple groups were analyzed by oneway ANOVA, with Tukey’s post hoc test conducted. The data in Supplementary Figure 1 B at
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different time points were analyzed by repeated measures ANOVA, and the Bonferroni’s post hoc
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Conflicts of Interest
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test was conducted. Each experiment was repeated 3 times.
The authors declare no conflict of interest.
Funding None.
Credit Author Statement Conceptualization: Li Yu, Ning Han; Data curation: Li Yu; Formal analysis: Songtian Che; Investigation: Li Yu; Methodology: Yazhen Wu; Project administration: Ning Han; Resources: Songtian Che; Software: Yazhen Wu; Supervision: Ning Han; Validation: Li Yu, Shuai Wu; 17
Journal Pre-proof Visualization: Shuai Wu; Roles/Writing - original draft: Li Yu, Ning Han; Writing - review & editing: Li Yu, Shuai Wu, Songtian Che, Yazhen Wu, Ning Han
References
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Figure 1 Poor expression of miR-203 and high expression of SNAI2 are found in mice and HRMECs with pathological retinal neovascularization disease. A, the vascular endothelial nucleus in neovascularization that pierced the retinal ILM measured by HE staining (× 200, the red arrow is the endothelial cell nucleus). B, the miR-203 expression in the retinal tissues of OIR and normal mice detected by RT-qPCR. C, the miR-203 expression in hypoxia- induced HRMECs examined by RT-qPCR. D, the location of miR-203 in retinal tissues evaluated by In Situ Hybridization (× 400). 19
Journal Pre-proof E, the location of SNAI2 in retinal tissues evaluated by immunohistochemistry (× 400). * p < 0.05 vs. the normal mice. The data in figures were measurement data and expressed as mean ± standard deviation. The paired data between two groups conformed to normal distribution and homogeneity of variance were compared by paired t test, and the unpaired data between two groups conformed to normal distribution and homogeneity of variance were compared by unpaired t test. n = 8. Figure 2 Overexpressing miR-203 represses viability, migration and angiogenesis but promotes apoptosis of hypoxia- induced HRMECs. The hypoxia- induced HRMECs were treated with miR-
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203 mimic and mimic NC. A, the miR-203 expression in HRMECs detected by RT-qPCR. C, cell viability of HRMECs determined by CCK-8 assay. D, cell apoptosis of HRMECs measured by flow
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cytometry. E, cell migration of HRMECs detected by Transwell assay (× 200). F, angiogenesis in
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HRMECs measured by angiogenesis assay (× 100). G, the expression of Ki-67, Bax, MMP2/MMP-9, VEGF and CD34 s in HRMECs examined by western blot analysis. * p < 0.05 vs.
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hypoxia-induced HRMECs treated with mimic NC. The values in figures were measurement data
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and expressed as mean ± standard deviation. Data between two groups were compared by unpaired t test, and data in Figure 2C at different time points were analyzed by repeated measure s ANOVA
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with Bonferroni’s post hoc test conducted. Each experiment was repeated 3 times.
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Figure 3 miR-203 targets SNAI2. A, the potential binding site between miR-203 and SNAI2 predicted by TargetScan database. B, the luciferase activity of Wt-SNAI2-3'UTR and Mut-SNAI23'UTR after co-transfection with mimic NC or miR-203 mimic detected by dual- luciferase reporter assay; * p < 0.05 vs. the cells treated with mimic NC. C, the expression of SNAI2 in retina tissues of OIR and normal mice examined by western blot analysis; * p < 0.05 vs. the tissues or cells in OIR and normal mice. E, the expression of SNAI2 in HRMECs after treatment with miR-203 mimic or miR-203 inhibitor; * p < 0.05 vs. the cells treated with inhibitor NC, # p < 0.05 vs. the cells treated with mimic NC. The measurement data were expressed as mean ± standard deviation. Data between two groups were compared by unpaired t test. Each experiment was repeated 3 times.
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Journal Pre-proof Figure 4 Overexpression of miR-203 or silencing of SNAI2 inactivates the GSK-3β/β-catenin pathway and represses viability, migration and angiogenesis but induces apoptosis of hypoxiainduced HRMECs. Here, the hypoxia- induced HRMECs were transfected with mimic NC + sh-NC, miR-203 mimic + sh-NC, mimic NC + sh-SNAI2 or miR-203 mimic + sh-SNAI2. A, the expression of miR-203 and SNAI2 in HRMECs detected by RT-qPCR. B, the cell viability of HRMECs examined by CCK-8 assay. C, the cell apoptosis of HRMECs detected by flow cytometry. D, the cell migration of HRMECs detected by Transwell assay (× 200). E, angiogenesis in
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HRMECs examined by angiogenesis assay (× 100). F, the expression of Ki-67, Bax, MMP-2/MMP9, VEGF, CD34, phosphorylated GSK-3β and β-catenin in HRMECs measured by western blot
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analysis. * p < 0.05 vs. hypoxia- induced HRMECs treated with mimic NC + sh-NC; # p < 0.05 vs.
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hypoxia-induced HRMECs treated with miR-203 mimic + sh-NC. The measurement data were expressed as mean ± standard deviation. The data among multiple groups were analyzed by one-
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way ANOVA, with Tukey’s post hoc test conducted. The data in Figure 4 B at different time points
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were analyzed by repeated measures ANOVA, and the Bonferroni’s post hoc test was conducted. Each experiment was repeated 3 times.
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Figure 5 Overexpressing miR-203 or silencing SNAI2 suppresses viability and angiogenesis of the
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retinal tissues of mice with pathological retinal neovascularization disease via suppression of the GSK-3β/β-catenin pathway. The OIR mice were treated with mimic NC + sh-NC, miR-203 mimic + sh-NC, mimic NC + sh-SNAI2 or miR-203 mimic + sh-SNAI2. A, the expression of miR-203 and SNAI2 in retinal tissues detected by RT-qPCR. B, the vascular endothelial nucleus in neovascularization that pierced the retinal ILM and the cell viability within retinal ILM observed in retinal tissues by HE staining (× 200, the red arrow is the endothelial cell nucleus). C, the expression of VEGF and CD34 and MVD level in retinal tissues by immunohistochemistry (× 400). D, MVD level in retinal tissues. E, the expression of Ki-67, Bax, MMP-2/MMP-9, VEGF, CD34, phosphorylated GSK-3β and β-catenin in retinal tissues measured by western blot analysis. * p < 0.05 vs. OIR mice treated with mimic NC + sh-NC; # p < 0.05 vs. OIR mice treated with miR-203 21
Journal Pre-proof mimic + sh-NC. The measurement data were expressed as mean ± standard deviation. The data among multiple groups were analyzed by one-way ANOVA, with Tukey’s post hoc test conducted. Each experiment was repeated 3 times. Figure 6 miR-203 regulates the progression of pathological retinal neovascularization disease via the GSK-3β/β-catenin pathway by modulating SNAI2. miR-203 was poorly expressed but SNAI2 was highly expressed in the mouse model of pathological retinal neovascularization disease, and the targeting relationship between miR-203 and SNAI2 was identified. Additionally, overexpressing
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miR-203 suppressed angiogenesis of mice with pathological retinal neovascularization disease through the suppression of the GSK-3β/β-catenin pathway by inhibiting SNAI2.
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Table 1 Primer sequences for RT-qPCR Genes
Sequences
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5’-GCCTCCAAGAAGCCCAACTA-3’
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β-actin
5’-CGAACTGGACACACATACAGTG-3’
R: 5’-CTGAGGATCTCTGGTTGTGGT-3’ F:
SNAI2 (mouse)
5’-ACACTCCAGCTGGCGTGAAATGTTTAGGACCA-3’
R: 5’-CTCAACTGGTGTCGTGGA-3’ F:
SNAI2 (HRMECs)
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R: 5’-GCCGCGTGAAATGTTTAGG-3’ F:
miR-203 (mouse)
5’-GTGCAGGGTCCGAGGT-3’
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F: miR-203 (HRMECs)
R: 5’-GCCGACGATGTCCATACAGT-3’ F:
5'-CTCCATCCTGGCCTCGCTGT-3'
R: 5'-GCTGTCACCTTCACCGTTCC-3'
Notes: RT-qPCR, reverse transcription quantitative polymerase chain reaction; miR-203, microRNA-203; F, forward; R, reverse.
Highlights
miR-203 is poorly expressed in pathological retinal neovascularization disease. SNAI2 is highly expressed in pathological retinal neovascularization disease. miR-203 targets SNAI2 in pathological retinal neovascularization disease. miR-203/SNAI2 axis inhibits angiogenesis via GSK-3β/β-catenin pathway. This study provides novel insights for treating pathological retinal neovascularization.
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