c-Jun negative feedback loop inhibits ovarian cancer carcinogenesis and progression

Biomedicine & Pharmacotherapy 125 (2020) 109865

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

miR-6089/MYH9/β-catenin/c-Jun negative feedback loop inhibits ovarian cancer carcinogenesis and progression

T

Longyang Liua,b,**, Yingxia Ningc,*, Juanjuan Yid,*, Jianhuan Yuane, Weiyi Fanga, Zhongqiu Linf, Zhaoyang Zengg a

Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, 510315, China Southern Medical University, Guangzhou, 510000, China c Department of Gynaecology and Obstetrics, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510120, China d Department of Dermatovenereology, Foshan Women and Children Hospital, Guangdong, 528000 China e Department of Gynecology, The First People's Hospital of Huizhou City, Guangdong, 516000 China f Department of Gynecology Oncology, The Memorial Hospital of Sun Yat-sen University, Guangzhou, 510000 China g Department of Gynecology, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, 510315 China b

A R T I C LE I N FO

A B S T R A C T

Keywords: miR-6089 MYH9 Ovarian cancer

The pathogenesis of ovarian cancer remains to be elucidated. Our previous study demonstrated that myosin heavy chain 9 (MYH9) overexpression was associated with poor prognosis of epithelial ovarian cancer. However, the mechanism of MYH9 and its regulation by microRNA (miR) is not clear. The results of the present study demonstrated that miR-6089 was one of the microRNAs targeting MYH9, and miR-6089 overexpression suppressed ovarian cancer cell proliferation, migration, invasion and metastasis in vivo and in vitro. Mechanistic studies confirmed that miR-6089 directly targeted MYH9 to inactivate the Wnt/β-catenin signalling pathway and its downstream epithelial-to-mesenchymal transition (EMT), cell-cycle factors and c-Jun, whereas overexpression of MYH9 reversed the inhibitory effects of miR-6089 overexpression in ovarian cancer cells by upregulating the Wnt/β-catenin and its downstream EMT, cell-cycle factors and c-Jun. Interestingly, miR-6089 was transcriptionally inhibited by c-Jun, a transcription factor which could be induced by MYH9 via the Wnt/βcatenin pathway. Thus miR-6089/MYH9/β-catenin/c-Jun formed a negative feedback loop in ovarian cancer. In clinical samples, miR-6089 negatively correlated with MYH9 expression. Our study is the first to demonstrate that miR-6089 serves as a tumor-suppressive miRNA, and miR-6089/MYH9/β-catenin/c-Jun negative feedback loop inhibits ovarian cancer carcinogenesis and progression.

1. Introduction Ovarian cancer (OC) is the leading cause of mortality among female reproductive malignant cancers in China [1], and it is the second most common cause of gynecologic cancer-related death in women worldwide [2]. Globally there are 239,000 new cases and 152,000 deaths every year, making ovarian cancer the seventh most common cancer and the second most common cause of gynecologic cancer-related mortality [2]. Despite new therapeutic approaches sought to treat OC, the mortality has remained constantly high, and the 5-year prognosis of patients with OC remains poor [3]. The poor therapeutic effects occur due to high biological malignancy and invasive capacity of OC cells. Therefore, it is important to explore the mechanisms driving OC

initiation and progression, which may help identify effective targeted treatment methods and improve the prognosis of patients with OC. In our previous study [4], myosin heavy chain 9 (MYH9) was demonstrated to be upregulated in epithelial OC tissues compared with paratumor tissue, and high MYH9 expression was associated with poor survival in patients with OC. However, the detailed molecular mechanisms of the role of MYH9 in OC initiation and progression remain to be elucidated, and the mechanism of MYH9 regulation by microRNAs (miRNAs) has not been reported in OC. miRNAs are small non-coding RNAs which lead to target gene mRNA cleavage or translational repression by binding to the 3′-untranslated region (3′-UTR) of target gene mRNAs. miRNAs play an important role in a great deal of physiological and pathological cellular

⁎⁎

Corresponding author at: Southern Medical University, Guangzhou, 510000. Corresponding authors. E-mail addresses: [email protected] (L. Liu), [email protected] (Y. Ning), [email protected] (J. Yi), [email protected] (J. Yuan), [email protected] (W. Fang), [email protected] (Z. Lin), [email protected] (Z. Zeng). ⁎

https://doi.org/10.1016/j.biopha.2020.109865 Received 6 November 2019; Received in revised form 4 January 2020; Accepted 10 January 2020 0753-3322/ © 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Biomedicine & Pharmacotherapy 125 (2020) 109865

L. Liu, et al.

measured at 490 nm with a Universal Microplate Reader (Bio-Tek instruments, Inc., Winooski, VT, USA).

processes, including cell proliferation, migration, invasion, differentiation and metastasis [5–16]. Recently, an increasing number of miRNAs have been demonstrated to be involved in OC carcinogenesis and progression [17–30]. However, the biological role and molecular mechanisms of microRNA (miR)-6089, which is a newly discovered miRNA, in OC initiation and progression have not been reported. The present study identified that miR-6089 as a potential tumor suppressor directly targeted MYH9 to inactivate the Wnt/β-catenin pathway and its downstream genes associated with epithelial-to-mesenchymal transition (EMT), cell-cycle factors and c-Jun. Further, we observed that miR-6089 was inhibited by c-Jun, an oncogenic transcription factor as Wnt/β-catenin-downstream positive regulator [31], which in turn was induced by MYH9 expression and thus formed a negative feedback loop among miR-6089, MYH9, β-catenin, and c-Jun involving in OC carcinogenesis and progression.

2.5. -Ethynyl-2′-deoxyuridine (EdU) analysis Proliferating SKOV3 and OVCAR3 cells were examined using the Cell-Light EDU Apollo 488 or 567 In Vitro Imaging kit (Guangzhou Ribobio Co., Ltd.) according to the manufacturers’ protocols, respectively. Briefly, following incubation with 10 μM EdU more than 2 h at 37 °C with 5 % CO2, SKOV3 and OVCAR3 cells were fixed with 4 % paraformaldehyde at room temperature, permeabilized in 0.3 % Triton X-100 and stained with Apollo fluorescent dyes, respectively. A total of 5 μg/mL DAPI was used to stain the cell nuclei for 15 min. The number of EdU-positive cells was counted under a fluorescence microscope in five random fields. All assays were independently performed three times.

2. Methods 2.6. Transwell and Boyden assays

2.1. Cells and patients

Cell migration and invasion assays were performed using Transwell chambers (8 μm, 24-well insert; Corning Inc., Lowell, MA, USA). For Transwell assay, cells (1 × 105) in serum-free medium were added to the upper chamber, and medium containing 20 % FBS was added to the lower chamber. Cells were incubated for 12 h. For Boyden assay, diluted Matrigel (BD Biosciences) was used to pre-coat the insert membrane. 1 × 105 cells were cultured for 36 h under the same conditions. Finally, the cells that migrated or invaded into the lower chambers were fixed with methanol, stained with Giemsa or crystal violet and counted in six random fields.

Human ovarian adenocarcinoma cell lines SKOV3 and OVCAR3 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium (Gibco) supplemented with 20 % fetal bovine serum (FBS, HyClone) at 37 °C with 5 % CO2. In addition, OC and paired paratumor tissues were acquired from patients undergoing a surgical procedure at the Integrated Hospital of Traditional Chinese Medicine, Southern Medical University. The patients provided written informed consent prior to the study. This study was approved by the Ethics Committee of the Integrated Hospital of Traditional Chinese Medicine, Southern Medical University.

2.7. Wound-healing assay

2.2. Transfection

SKOV3 and OVCAR3 cells were seeded into six-well plates at 95 % confluence. A scratch wound was created using a sterile 10 μL pipette tip. And then the cells were cultured in serum-free RPMI 1640 medium. Phase-contrast images were captured at 0 and 48 h after the scratch. Three random fields were measured.

The miR-6089 mimic and siRNA targeting MYH9 (siMYH9) or c-Jun (si-c-Jun) were designed by Guangzhou Ribobio Co., Ltd. (Guangzhou, China). The miR-6089, siMYH9 and si-c-Jun [31] sequences are presented in Supplementary Table 1. In MYH9 and c-Jun overexpression experiments, plasmids encoding MYH9 and c-Jun were designed by Shanghai GeneChem Co., Ltd. (Shanghai, China). SiRNA or plasmids were transfected into SKOV3 and OVCAR3 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol.

2.8. Dual luciferase reporter assay This experiment was performed in 293 T cells to determine the binding between miR-6089 and MYH9 mRNA 3′-UTR as described in Figs. 1 and 2 A which was predicted by Targetscan software. Briefly, 293 T cells were transferred to a 24-well plate at a density of 1 × 105 cells/well for 24 h prior to the experiment. A total of 50 ng luciferase reporter vectors and 150 ng siMYH9 or miR-6089 mimic were cotransfected into 293 T cells. Cells were incubated for 2 days and harvested for luciferase activity measurement. The Promega Corporation system was used for this measurement. Renilla and firefly luciferase activities were measured, and the ratio of firefly and Renilla luciferase intensity was calculated.

2.3. Reverse transcription-quantitative polymerase chain reaction (RTqPCR) RNA was extracted from clinical fresh tissues and cell lines using QIAZOL (Qiagen, Shanghai, China). Complementary DNA (cDNA) was synthesized from 1 μg extracted RNA using random primers and Maxima First Strand cDNA Synthesis Kit (Takara Bio, Inc., Otsu, Japan). SYBR® Green Master Mix was used for qPCR. The primers are presented in Supplementary Table 2. All primers were validated using the standard PCR method. GAPDH used as the reference gene for MYH9 or cJun; U6 was used as the reference gene for miRNAs. The relative expression of RNAs was calculated using formula [31].

2.9. Chromatin immunoprecipitation (ChIP) According to the manufacturer’s instructions, ChIP assay kit (cat. no. 17-371; EMD Millipore) was used to examine whether c-Jun bound to the miR-6089 promoter as predicted by PROMO and JASPAR software. SKOV3 and OVCAR3 cells were fixed with 1 % formaldehyde to covalently crosslink proteins to DNA, and chromatin was harvested from the cells. Crosslinked DNA was sheared to 200-1,000 base pairs by sonication and subjected to an immunoselection process using anti-cJun and normal IgG antibodies (Supplementary Table 4). Finally, PCR was used to determine the enrichment of DNA fragments in the putative c-Jun binding sites in the miR-6089 promoter using specific primers presented in Supplementary Table 3.

2.4. -(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay MTT was used to detect the effect of miR-6089 and MYH9 expression on OC cell viability at different time points (1, 2, 3 and 4 days after miR-6089 mimic or siMYH9 transfection). Briefly, 2 × 103 cells were incubated with MTT for at least 4 h to produce formazan. Then the formazan crystals formed by viable cells were solubilized in 150 μL dimethyl sulfoxide (Sigma). At last, the absorbance value (OD) was 2

Biomedicine & Pharmacotherapy 125 (2020) 109865

L. Liu, et al.

Fig. 1. miR-367-3p, miR-6089, MYH9 and c-Jun mRNA expression in fresh OC and paired paratumor tissues. A. miR-367-3p mRNA expression level in fresh OC tissues was lower than that in paired paratumor tissues. B. miR-6089 mRNA expression level in fresh OC tissues was lower than that in paratumor tissues. C. MYH9 mRNA expression level in fresh OC tissues was higher than that in paratumor tissues. D. c-Jun mRNA expression level in fresh OC tissues was higher than that in paratumor tissues. E. miR-6089 mRNA expression level in 16 cases of fresh OC tissues and paired paratumor tissues (all of paratumor tissues’ expression level were set as 1.00 ± 0.00). F. MYH9 mRNA expression level in 16 cases of fresh OC tissues and paired paratumor tissues (all of paratumor tissues’ expression level were set as 1.00 ± 0.00). G. c-Jun mRNA expression level in 16 cases of fresh OC tissues and paired paratumor tissues (all of paratumor tissues’ expression level were set as 1.00 ± 0.00). H. Pearson correlation analysis revealed the significantly inverse correlation between miR-6089 and MYH9 levels in fresh OC tissues. I. Pearson correlation analysis revealed the significantly inverse correlation between miR-6089 and c-Jun levels in fresh OC tissues.

Myc, MYH9, cyclin D1 (CCND1), and GAPDH (Supplementary Table 4) overnight at 4 °C. Following washing three times with TBST, the membrane was incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibody at 37 °C for 1 h. The membranes were visualized using an electrochemiluminescence chromogenic kit (Beyotime Institute of Biotechnology) in a dark room and images were captured with a ChemiDoc™ Molecular Imager (Bio-Rad Laboratories, Inc.). The experiments were repeated at least three times.

2.10. Western blotting The flasks with ovarian cancer cells were washed three times with ice-cold PBS and total protein was harvested using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing PMSF (Bio-Rad Laboratories, Inc.) and Phosphatase inhibitors (Bio-Rad Laboratories, Inc.) (100:1:1). A bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) was used to determine the protein concentrations. Proteins (30 μg) were separated by SDS-PAGE on 10 % gels and transferred to a PVDF membrane (Beyotime Institute of Biotechnology). The membranes were blocked with 5 % bovine serum albumin in TBS containing Tween-20 (TBST) for 1 h at 37 °C and incubated with primary antibodies against N-cadherin, E-cadherin, Vimentin, β-catenin, Transcription factor 4 (TCF4), c-Jun, β-tubulin, c-

2.11. Lentivirus production and infection Lentiviral particles carrying the hsa-miR-6089 precursor and negative control vectors were both constructed by GeneChem (Shanghai, China). SKOV3 and OVCAR3 cells were infected with miR-6089 3

Biomedicine & Pharmacotherapy 125 (2020) 109865

L. Liu, et al.

Fig. 2. miR-6089 inhibits ovarian cancer cell proliferation, migration, invasion and metastasis via the Wnt/β-catenin pathway. miR-6089 mimics suppressed cell proliferation identified using MTT assay in ovarian cancer SKOV3 and OVCAR3 cells. B. miR-6089 mimics reduced cell proliferation identified using EdU in ovarian cancer cells (x200 magnification; scale bar, 50 μm). C. miR-6089 mimics reduced cell migration and invasion identified using Transwell and Boyden assays in ovarian cancer cells (x200 magnification; scale bar, 50 μm). D. miR-6089 mimics reduced cell migration identified using woundhealing assay in ovarian cancer cells (x40 magnification; scale bar, 250 μm). E. Subcutaneous tumor formation in nude mice injected with miR-6089-overexpression or NC cells in the right and left flank, respectively (n = 5). F. The tumor volume generated by the miR-6089-overexpressing and NC ovarian cancer cells. G. The mean tumor weight generated by the miR-6089-overexpressing and NC ovarian cancer cells. H. The tumor formation in the lungs of nude mice injected with miR-6089overexpressing or NC cell. I. miR-6089 mimics reduced the expression of the Wnt/β-catenin pathway and its downstream factors in ovarian cancer.

overexpressing or negative control (NC) vectors, respectively. Green fluorescent protein ratio was used to determine the infection efficiency. Overexpression efficiency of miR-6089 was detected by RT-qPCR.

2.12. Animal studies To evaluate the effect of miR-6089 in vivo, SKOV3 and OVCAR3 cells were infected with a lentivirus overexpressing miR-6089 or the 4

Biomedicine & Pharmacotherapy 125 (2020) 109865

L. Liu, et al.

Fig. 3. miR-6089 directly targets MYH9. Schematic model of the binding site for miR-6089 in the 3′-untranslated region (3′-UTR) of the MYH9 mRNA using Targetscan. B. Western blotting analysis of MYH9 protein levels in SKOV3 and OVCAR3 cells after transfection with the miR-6089 mimic. C. Reverse transcription-quantitative PCR (RT-qPCR) analysis of MYH9 levels in SKOV3 and OVCAR3 cells transfected with the miR-6089 mimic and control. D. The relative luciferase activity in 293 T cells transfected with wild-type or mutant MYH9 3′-UTR.

The differences between two groups were analyzed by Student’s t-test. Two-tailed Pearson correlation analysis was performed and graphs were made using GraphPad Prism 7 (GraphPad Software, Inc.). Throughout the text, figures and figure legends, the following symbols were used to denote statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001.

negative control (GeneChem, Shanghai, China). 4-week-old female nude mice (BALB/C, female) were purchased from the Experimental Animal Center of Southern Medical University (Guangzhou, China) and maintained under pathogen-free conditions. The miR-6089-overexpressing and miR-NC SKOV3 or OVCAR3 cells were harvested and resuspended in PBS. For the subcutaneous xenograft tumor studies, a total of 2.5 × 106 SKOV3 or OVCAR3 cells in 100 μl PBS were subcutaneously injected into the rear flank of the mice (left side, negative control; right side, miR-6089). Tumors were measured using a caliper, and tumor volume was calculated as follows: V = L × W2 × 0.5236, where L is the length and W is the width of the tumor. For the lung xenograft tumor studies, a total of 5 × 105 SKOV3 or OVCAR3 cells in 100 μl PBS were injected into the tail vein of the mice. After 30 days, the mice were euthanized with Chloral hydrate, and the tumors were harvested and weighed. All animals were maintained and treated in accordance with the guidelines of the Institutional Animal Care and Use Committee of Southern Medical University.

3. Results 3.1. miR-6089 was predicted as a candidate regulating factor of MYH9 In our previous study [4], oncogenic MYH9 was demonstrated to be closely associated with the progression and prognosis of patients with OC. To explore the mechanism of MYH9 in OC, TargetScan software was used to predict the miRNAs that potentially target MYH9. In addition, RT-qPCR was used to screen the candidate miRNAs. Due to the inverse expression pattern between miRNAs and their target mRNA, miR-367-3p and miR-6089 were identified to be downregulated in fresh OC tissues compared with paratumor tissues (Fig. 1 A, B, D and Supplementary Table 5). In 16 cases of patients, miR-6089 was upregulated in patient 3 and 11, which may due to their lower differentiation grade and lower clinical stage. As miR-6089 has rarely been reported in human cancer, it was selected for further analysis.

2.13. Immunohistochemical staining (IHC) Paraffin sections prepared from in vivo mouse experiments were used for immunohistochemistry assays to detect the protein expression of Ki67 and PCNA. The indirect streptavidin-peroxidase method was used according to the manufacturer’s introduction. Immunohistochemically stained sections were examined separately by two professional pathologists. The antibodies are presented in Supplementary Table 4.

3.2. miR-6089 inhibits OC cell proliferation, migration, invasion and metastasis in vitro and in vivo To further explore the role and mechanism of miR-6089 in OC cells, a miR-6089 mimic was used to transfect OC SKOV3 and OVCAR3 cells. MTT and EdU assays revealed that the miR-6089 mimic significantly decreased the proliferation of SKOV3 and OVCAR3 cells (Fig. 2A, B). In addition, Transwell, Boyden and wound-healing assays demonstrated

2.14. Statistical analysis Each experiment was performed at least three times. The data are presented as the mean ± SD of at least three independent experiments. 5

Biomedicine & Pharmacotherapy 125 (2020) 109865

L. Liu, et al.

by suppressing MYH9 via Wnt/β-catenin signalling pathway.

that the migratory and invasive abilities were significantly inhibited by the miR-6089 mimic (Fig. 2C, D). In addition, to further study whether miR-6089 affected tumor growth and lung metastasis, subcutaneous and tail vein injected transplantation tumor experiments were performed in nude mice. The results revealed that miR-6089 suppressed the proliferation (Fig. 2F) and metastasis(Fig. 2E) of the tumor in vivo, and that mice injected with miR-6089-overexpressing (the overexpressing effects are presented in Supplementary Fig. 1C) cells exhibited a lower number of tumor nodules and formed smaller tumors compared with the controls (Fig. 2F, G, H). In addition, western blotting assays demonstrated that the Wnt/β-catenin signalling pathway and its downstream EMT and cell-cycle factors were significantly inhibited (Fig. 2I). These results suggested that miR-6089 inhibited the proliferation, invasion and metastasis of OC in vitro and in vivovia the Wnt/ β-catenin pathway.

3.6. miR-6089 is directly inhibited by c-Jun To further explore the mechanism of miR-6089 regulation by transcription factors in OC cells, the putative transcription factor binding sites in the miR-6089 promoter were predicted. c-Jun, which can be induced by MYH9 (Fig. 4E and Supplementary Fig. 1B), was one of the candidate transcription factors that could bind to the miR-6089 promoter (Fig. 6A). Next, overexpression of c-Jun in SKOV3 and OVCAR3 cells (the overexpressing effects are presented in Supplementary Fig. 1D) resulted in lower miR-6089 mRNA expression compared with that in the control cells (Fig. 6B), and knockdown of c-Jun in SKOV3 and OVCAR3 cells (the interfering effects are presented in Supplementary Fig. 1E) resulted in higher miR-6089 mRNA expression compared with that in the control cells (Fig. 6C). Further, the ChIP assay in SKOV3 and OVCAR3 cells revealed that c-Jun bound at sites 1, 2 and 3 in the miR-6089 promoter (Fig. 6D). These results suggested that miR6089 was directly inhibited by c-Jun.

3.3. miR-6089 directly targets MYH9 To further demonstrate that miR-6089 directly targeted MYH9, TargetScan software was used to predict the binding sites of miR-6089 in the MYH9 3′-UTR (Fig. 3A). Then, the effects of miR-6089 on MYH9 protein expression were investigated. MYH9 protein expression in SKOV3 and OVCAR3 cells was significantly inhibited by miR-6089 overexpression (Fig. 3B). However, MYH9 mRNA expression in SKOV3 and OVCAR3 cells was not inhibited by miR-6089 overexpression (Fig. 3C). Further, dual luciferase reporter assay was performed to demonstrate the direct interaction. The luciferase reporter assay demonstrated that compared with the control group, overexpression of miR6089 inhibited the luciferase activity (Fig. 3D). Point mutations were introduced into sites complementary to miR-6089 within the MYH9 3′UTR (Fig. 3A); the results demonstrated that the luciferase reporter was not affected by miR-6089 overexpression or knockdown (Fig. 3D). These results suggested that miR-6089 directly targeted MYH9 at the post-transcriptional level.

3.7. miR-6089 reverses the effects of c-Jun on cell proliferation, migration and invasion in OC cells To determine whether miR-6089 reversed c-Jun expression and thus affected cell functions, MTT and EdU assays were performed (Fig. 7A, B). In addition, Transwell, Boyden and wound-healing assays were performed to explore the reverse effect of miR-6089 on c-Jun-induced effects on OC cell migration and invasion (Fig. 7C, D), and western blotting was performed to explore the mechanism of these effects (Fig. 7E). Overexpression of miR-6089 reversed the c-Jun-induced promotion of cell proliferation, migration and invasion, and western blotting demonstrated that overexpression of miR-6089 reversed the cJun-induced Wnt/β-catenin signalling. Altogether, these results demonstrated that miR-6089 regulated by c-Jun inhibited OC cell proliferation, migration and invasion.

3.4. MYH9 knockdown inhibits OC cell proliferation, migration and invasion via the Wnt/β-catenin pathway

3.8. miR-6089, MYH9 and c-Jun expression in fresh OC and paratumor tissues

To further explore the role and mechanism of MYH9 in OC cells, siMYH9 was used to transfect SKOV3 and OVCAR3 cells (the interfering effects are presented in Supplementary Fig. 1A). MTT and EdU assays demonstrated that siMYH9 significantly decreased the proliferation of SKOV3 and OVCAR3 cells (Fig. 4A, B). In addition, Transwell, Boyden and wound-healing assays indicated that the migratory and invasive abilities of the cells were significantly inhibited by siMYH9 (Fig. 4C, D). In addition, western blotting demonstrated that the Wnt/β-catenin signalling pathway and its downstream factors (including EMT and cellcycle factors and c-Jun) were significantly inhibited (Fig. 4E). Therefore, MYH9 promoted the proliferation, migration and invasion of OC in vitrovia the Wnt/β-catenin pathway.

Our previous study [4] demonstrated that MYH9 was upregulated in paraffin-embedded OC tissues compared with paratumor tissues and that MYH9 was an indeed independent prognostic indicator of OC. The results of this study revealed that MYH9 and c-Jun mRNA expression level in fresh OC tissues were both significantly higher compared with that in paratumor tissues (Fig. 1C, D, F, G and Supplementary Table 6, 7). By contrast, miR-6089 mRNA expression level in fresh OC tissues was significantly lower compared with that in paratumor tissues (Fig. 1B, E and Supplementary Table 5). The relationship among MYH9, miR-6089 and c-Jun mRNA expression was analyzed by Pearson correlation analysis, which revealed a significant negative correlation between miR-6089 and MYH9 levels in fresh OC tissues (Fig. 1H), and also a significant negative correlation between miR-6089 and c-Jun levels (Fig. 1I).

3.5. MYH9 reverses the effects of miR-6089 on cell proliferation, migration and invasion in OC cells

4. Discussion

To determine whether MYH9 reverses miR-6089 expression to affect cell functions, MTT and EdU assays were performed to determine the effects of MYH9 on the miR-6089-inhibited OC cell proliferation (Fig. 5A, B). In addition, Transwell, Boyden and wound-healing assays were performed to explore the reverse effects of MYH9 on miR-6089inhibited OC cell migration and invasion (Fig. 5C, D). Western blotting was performed to explore the underlying mechanism. Overexpression of MYH9 reversed the miR-6089-mediated inhibition of cell proliferation, migration and invasion, and western blotting revealed that overexpression of MYH9 reversed the miR-6089-mediated inhibition of the Wnt/β-catenin signalling pathway (Fig. 5E). These data demonstrated that miR-6089 inhibited OC cell proliferation, migration and invasion

Our previous study [4] demonstrated that MYH9 was upregulated and associated with poor prognosis in epithelial ovarian cancer, but the regulatory mechanism of MYH9 in OC is unknown. The results of this study revealed that miR-6089 was newly predicted as a candidate regulating factor of MYH9, and further demonstrated that miR-6089 inhibited OC cell proliferation, migration, invasion and metastasis in vitro and in vivovia the Wnt/β-catenin signalling pathway and its downstream EMT and cell-cycle factors. The Wnt/β-catenin pathway and its downstream factors including EMT and cell-cycle factors have been demonstrated to be the crucial signal in the proliferation, 6

Biomedicine & Pharmacotherapy 125 (2020) 109865

L. Liu, et al.

Fig. 4. Knocking down MYH9 suppresses ovarian cancer cell proliferation, migration and invasion via the Wnt/β-catenin pathway. Knocking down MYH9 suppressed cell proliferation identified by MTT assay in ovarian cancer SKOV3 and OVCAR3 cells. B. Knocking down MYH9 reduced cell proliferation identified by EdU in ovarian cancer cells (x200 magnification; scale bar, 50 μm). C. Knocking down MYH9 reduced cell migration and invasion identified by Transwell and Boyden assays in ovarian cancer cells (x200 magnification; scale bar, 50 μm). D. Knocking down MYH9 reduced cell migration identified using wound-healing assay in ovarian cancer cells (x40 magnification; scale bar, 250 μm). E. Suppression of MYH9 by siRNA reduced the expression of the Wnt/βcatenin pathway and downstream factors in ovarian cancer.

role in OC cell proliferation, invasion and metastasis. Transcription factors regulate miRNAs via combining with the binding sites in their promoter regions [53–58]. To further explore the upstream regulation of miR-6089, PROMO and JASPAR software were used to predict the transcription factors which may bind to the promoter of miR-6089. Interestingly, c-Jun was predicted as one of the candidate transcription factors. Subsequent study showed that overexpression of c-Jun could inhibit miR-6089 expression, while knockdown of c-Jun could promote miR-6089 expression. Further, ChIP assay was used to confirm the binding sites of c-Jun, which demonstrated that c-Jun could bind to the promoter of miR-6089. And the complementary assays demonstrated that miR-6089 overexpression suppressed the cJun-induced OC cell proliferation, migration and invasion. As we all know, c-Jun is a classic transcription factor in human cancers that regulates various functions in multiple cells, including cell proliferation, migration, invasion, metastasis and death [31,53–61], and it is a Wnt/β-catenin-downstream positive regulator [31]. In this study, we observed that c-Jun could be induced by MYH9 via Wnt/β-catenin and its downstream factors, which is similar to our previous study [31,] that MYH9 increases β-catenin expression and promotes glycogen synthase kinase 3β protein degradation by activating the PI3K/AKT/c-Jun signalling pathway in nasopharyngeal carcinoma. Therefore, this study showed that MYH9 could induce c-Jun expression to inhibit miR-6089.

migration, invasion and metastasis of various cancers [32–41]. Thus, these results suggested that miR-6089 may function as a tumor suppressor in OC cells. Furthermore, we observed that miR-6089 directly targeted MYH9 using Dual luciferase reporter assay, which was consistent with Targetscan bioinformatics prediction. In previous study, MYH9 played a dual role in cancer cell proliferation, survival, invasion and metastasis [42–49]. Earlier reports showed that MYH9 serves a tumor suppressor role in skin [46], head and neck squamous cell [47], tongue squamous cell [48] cancers and human invasive lobular breast carcinoma [49]. Inversely, recent reports show that MYH9 serves an oncogenic role, such as gastric [42,45], colorectal [43], esophageal squamous cell [50], non-small cell lung [51], breast [52] cancers, nasopharyngeal carcinoma [31], and so on. In this study we demonstrated for the first time that MYH9 serves an oncogenic role in OC and promotes OC cell proliferation, migration and invasion via the Wnt/β-catenin and its downstream EMT, cell-cycle factors and c-Jun, which supported our previous report [4] that oncogenic MYH9 was associated with ovrian cancer progression. Further, the complementary experiments in the present study demonstrated that the inhibitory effects of miR-6089 could be reversed by MYH9 overexpression via the Wnt/β-catenin and its downstream EMT, cell-cycle factors, and c-Jun. These results showed that miR-6089/MYH9/β-catenin/c-Jun signalling served an important 7

Biomedicine & Pharmacotherapy 125 (2020) 109865

L. Liu, et al.

Fig. 5. MYH9 reverses the effects of miR-6089 on cell proliferation, migration and invasion in ovarian cancer cells. A. MYH9 reverses the effects of miR-6089 on ovarian cancer cell proliferation identified using MTT assay. B. MYH9 reverses the effects of miR-6089 on ovarian cancer cell proliferation identified using EdU assay. C. MYH9 reverses the effects of miR-6089 on ovarian cancer cell migration and invasion identified using Transwell and Boyden assays. D. MYH9 reverses the effects of miR-6089 on ovarian cancer cell migration identified using wound-healing assay. E. Western blotting was used to determine the mechanism of the reverse effect; overexpression of MYH9 reversed the effects of miR-6089 on the Wnt/β-catenin signalling pathway.

Fig. 6. miR-6089 is directly inhibited by c-Jun. Schematic representation of the promoter regions of primary miR-6089 with the putative c-Jun transcription factor binding sites using PROMO and JASPAR. B. miR6089 expression levels were detected by RT-qPCR in ov-c-Jun (c-Jun overexpressing) ovarian cancer cells. C. miR-6089 expression levels were detected by RT-qPCR in si-c-Jun ovarian cancer cells. D. Chromatin immunoprecipitation analysis of c-Jun binding to the miR-6089 promoter region in ovarian cancer cells.

8

Biomedicine & Pharmacotherapy 125 (2020) 109865

L. Liu, et al.

Fig. 7. miR-6089 reverses the effects of c-Jun on ovarian cancer cell proliferation, migration and invasion. miR-6089 reverses the proliferative effects of c-Jun identified using MTT assay in ovarian cancer cells. B. miR-6089 reverses the proliferative effects of c-Jun identified using EdU in ovarian cancer cells. C. miR-6089 reverses the migratory effects of c-Jun identified using Transwell and Boyden assays in ovarian cancer cells. D. miR-6089 reverses the migratory effects of c-Jun identified using wound-healing assay in ovarian cancer cells. E. Western blotting was used to identify the mechanism of the reverse effects. Overexpression of miR-6089 reversed the c-Jun-induced promotion of the Wnt/β-catenin signalling pathway.

in this study were according with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was provided and signed by all patients prior to sample collection. All animal experiments were conducted strictly according with the recommendations in the Guide for the Care and Use of Laboratory Animals of Southern Medical University.

Thus, miR-6089/MYH9/β-catenin/c-Jun formed a negative feedback loop, which could inhibit OC cell proliferation, migration, invasion and metastasis. When the feedback loop is imbalanced, such as when miR6089 is downregulated, the feedback loop may allow OC cells to become more malignant and metastasize more rapidly. Therefore, the present study identified a new mechanism of OC carcinogenesis and progression, which may serve as a target for OC treatment. In addition, in clinical samples, low miR-6089 expression was negatively correlated with MYH9 expression. Taken together, the results reveal that miR-6089 serves a tumorsuppressive miRNA in OC, which directly targets MYH9 to inactivate the Wnt/β-catenin and its downstream EMT, cell-cycle factors and cJun. Interestingly, it is negatively regulated by c-Jun, an oncogenic transcription factor induced by MYH9 via Wnt/β-catenin and its downstream factors. Thus, miR-6089/MYH9/β-catenin/c-Jun forms a negative feedback loop, which inhibits OC cell proliferation, migration, invasion and metastasis. Accordingly, miR-6089 may be a novel therapeutic target for OC.

7. Consent for publication Not applicable. 8. Availability of data and material The datasets generated/analysed during the present study are available. Authors' contributions

5. Conclusion LLY performed the experiments and writed the original draft, NYX participated in the experiments and provided the funding acquisition, YJJ and YJH participated in the experiments and did the data curation with software, LZQ participated in the experiments and provided the resources, ZZY and FWY conceived, designed and supervised the experiments. All authors read and approved the final manuscript.

In conclusion, the present study identified a negative feedback loop of miR-6089/MYH9/β-catenin/c-Jun in OC that may inhibit OC carcinogenesis and progression. 6. Ethics approval and consent to participate

Declaration of Competing Interest

The Ethics Committee of The Integrated Hospital of Traditional Chinese Medicine, Southern Medical University authorized the experimental and research protocols of this study. All procedures performed

The authors declare no potential conflicts of interest. 9

Biomedicine & Pharmacotherapy 125 (2020) 109865

L. Liu, et al.

Acknowledgements

Cancer, Biomed Res. Int. 2014 (2014) 249393. [22] K.M. Elias KM, W. Fendler, K. Stawiski, S.J. Fiascone, A.F. Vitonis, R.S. Berkowitz, G. Frendl, P. Konstantinopoulos, C.P. Crum, M. Kedzierska, D.W. Cramer, D. Chowdhury, Diagnostic potential for a serum miRNA neural network for detection of ovarian cancer, eLife 6 (2017) e28932. [23] F. Wang, J.T. Chang, C.J. Kao, R.S. Huang, High expression of miR-532-5p, a tumor suppressor, leads to better prognosis in ovarian cancer both in vivo and in vitro, Mol. Cancer Ther. 15 (2016) 1123–1131. [24] L. Zhang, S. Volinia, T. Bonome, G.A. Calin, J. Greshock, N. Yang, C.G. Liu, A. Giannakakis, P. Alexiou, K. Hasegawa, C.N. Johnstone, M.S. Megraw, S. Adams, H. Lassus, Huang J, S. Kaur, S. Liang, P. Sethupathy, A. Leminen, V.A. Simossis, R. Sandaltzopoulos, Y. Naomoto, D. Katsaros, P.A. Gimotty, A. DeMichele, Q. Huang, R. Bützow, A.K. Rustgi, B.L. Weber, M.J. Birrer, A.G. Hatzigeorgiou, C.M. Croce, G. Coukos, Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer, Proc. Natl. Acad. Sci. U S A 105 (2008) 7004–7009. [25] L. Zhao, W. Wang, L. Xu, T. Yi, X. Zhao, Y. Wei, L. Vermeulen, A. Goel, S. Zhou, X. Wang, Integrative network biology analysis identifies miR-508-3p as the determinant for the mesenchymal identity and a strong prognostic biomarker of ovarian cancer, Oncogene 38 (2019) 2305–2319. [26] P.W. Choi, S.W. Ng, The functions of MicroRNA-200 family in ovarian Cancer: beyond epithelial-mesenchymal transition, Int. J. Mol. Sci. 18 (2017) 1207. [27] H. Yan, H. Li, P. Li, X. Li, J. Lin, L. Zhu, M.A. Silva, X. Wang, P. Wang, Z. Zhang, Long noncoding RNA MLK7-AS1 promotes ovarian cancer cells progression by modulating miR-375/YAP1 axis, J. Exp. Clin. Cancer Res. 37 (2018) 237. [28] J. Yan, Y. Jia, H. Chen, W. Chen, X. Zhou, Long non-coding RNA PXN-AS1 suppresses pancreatic cancer progression by acting as a competing endogenous RNA of miR-3064 to upregulate PIP4K2B expression, J. Exp. Clin. Cancer Res. 38 (2019) 390. [29] H. Zhu, X. Gan, X. Jiang, S. Diao, H. Wu, J. Hu, ALKBH5 inhibited autophagy of epithelial ovarian cancer through miR-7 and BCL-2, J. Exp. Clin. Cancer Res. 38 (2019) 163. [30] S. Dong, R. Wang, H. Wang, Q. Ding, X. Zhou, J. Wang, K. Zhang, Y. Long, S. Lu, T. Hong, H. Ren, K. Wong, X. Sheng, Y. Wang, Y. Zeng, HOXD-AS1 promotes the epithelial to mesenchymal transition of ovarian cancercells by regulating miR-1865p and PIK3R3, J. Exp. Clin. Cancer Res. 38 (2019) 110. [31] Y. Liu, Q. Jiang, X. Liu, X. Lin, Z. Tang, C. Liu, J. Zhou, M. Zhao, X. Li, Z. Cheng, L. Li, Y. Xie, Z. Liu, W. Fang, Cinobufotalin powerfully reversed EBV-miR-BART22induced cisplatin resistance via stimulating MAP2K4 to antagonize non-muscle myosin heavy chain IIA/glycogen synthase 3β/β-catenin signaling pathway, EBioMedicine 19 (2019) S2352–3964. [32] L.N. Zhang, Y.H. Huang, L. Zhao, Fusion of macrophages promotes breast cancer cell proliferation, migration and invasion through activating epithelial-mesenchymal transition and Wnt/β-catenin signaling pathway, Arch. Biochem. Biophys. 676 (2019) 108137. [33] J.H. Hwang, J. Yoon, Y.H. Cho, P.H. Cha, J.C. Park, K.Y. Choi, A mutant KRASinduced factor REG4 promotes Cancer stem cell properties via wnt/β-catenin signaling, Int. J. Cancer 12 (2019) 32728. [34] Q. Liu, Z. Wang, X. Zhou, M. Tang, W. Tan, T. Sun, Y. Deng, miR-342-5p inhibits osteosarcoma cell growth, migration, invasion, and sensitivity to Doxorubicin through targeting Wnt7b, Cell Cycle 10 (2019) 1–12. [35] Y.K. Ma, T.H. Shen, X.Y. Yang, Upregulation of LncRNA FAM83H-AS1 in hepatocellular carcinoma promotes cell proliferation, migration and invasion by Wnt/βcatenin pathway, Eur. Rev. Med. Pharmacol. Sci. 23 (2019) 7855–7862. [36] Q. Zhou, S. Chen, M. Lu, Y. Luo, G. Wang, Y. Xiao, L. Ju, X. Wang, EFEMP2 suppresses epithelial-mesenchymal transition via Wnt/β-catenin signaling pathway in human bladder cancer, Int. J. Biol. Sci. 15 (2019) 2139–2155. [37] I. Pinto, M. Duque, J. Gonçalves, P. Akkapeddi, M.L. Oliveira, R. Cabrita, J.A. Yunes, S.K. Durum, J.T. Barata, R. Fragoso, NRARP displays either pro- or antitumoral roles in T-cell acute lymphoblastic leukemia depending on Notch and Wnt signaling, Oncogene 4 (2019) s41388. [38] J. Shan, M.A. Al-Muftah, M.K. Al-Kowari, S.W.J. Abuaqel, K. Al-Rumaihi, I. AlBozom, P. Li, L. Chouchane, Targeting Wnt/EZH2/microRNA-708 signaling pathway inhibits neuroendocrine differentiation in prostate cancer, Cell Death Discov. 5 (2019) s41420. [39] H.V. Shaw, A. Koval, V.L. Katanaev, Targeting the Wnt signalling pathway in cancer: prospects and perils, Swiss Med. 149 (2019) w20129. [40] Y. Ji, H. Hao, K. Reynolds, M. McMahon, C.J. Zhou, Wnt signaling in neural crest ontogenesis and oncogenesis, Cells 8 (2019) E1173. [41] N. Mavila, J. Thundimadathil, The emerging roles of Cancer stem cells and Wnt/ Beta-Catenin signaling in Hepatoblastoma, Cancers (Basel) 11 (2019) E1406. [42] B. Yang, A. Wu, Y. Hu, C. Tao, J.M. Wang, Y. Lu, R. Xing, Mucin 17 inhibits the progression of human gastric cancer by limiting inflammatory responses through a MYH9-p53-RhoA regulatory feedback loop, J. Exp. Clin. Cancer Res. 38 (2019) 283. [43] B. Wang, X. Qi, J. Liu, R. Zhou, C. Lin, J. Shangguan, Z. Zhang, L. Zhao, G. Li, MYH9 promotes growth and metastasis via activation of MAPK/AKT signaling in colorectal Cancer, J. Cancer 10 (2019) 874–884. [44] Y. Wang, S. Liu, Y. Zhang, J. Yang, Myosin heavy chain 9: oncogene or tumor suppressor gene? Med. Sci. Monit. 25 (2019) 888–892. [45] G. Ye, K. Huang, J. Yu, L. Zhao, X. Zhu, Q. Yang, W. Li, Y. Jiang, B. Zhuang, H. Liu, Z. Shen, D. Wang, L. Yan, L. Zhang, H. Zhou, Y. Hu, H. Deng, H. Liu, G. Li, X. Qi, Erratum: MicroRNA-647 targets SRF-MYH9 axis to suppress invasion and metastasis of gastric cancer: erratum, Theranostics 8 (2018) 6350–6351. [46] D. Schramek, A. Sendoel, J.P. Segal, S. Beronja, E. Heller, D. Oristian, B. Reva, E. Fuchs, Direct in vivo RNAi screen unveils myosin IIa as a tumor suppressor of squamous cell carcinomas, Science 343 (2014) 309–313.

We gratefully thank Pro Zhaoyang Zeng for her help in patients’ samples collection of ovarian cancer. Funding This work was supported by the Guangdong Provincial Medical Research Fund (grant no. A2019096), the Guangdong Provincial Traditional Chinese Medicine Bureau Fund (grant no. 20201220), the President funds of Integrated Hospital of Traditional Chinese Medicine, Southern Medical University (No. 1201902001; No. 1201901002), the High-Level Academic Talent Training Program of Guangzhou Medical University (No. B185004083; No. B195001100), Guangzhou Science and Information Bureau Item (grant no. 201904010013), Natural Science Foundation of Guangdong Province (grant no. 2018A0303130180) and Hospital achievement transformation and Cultivation Project (grant no. ZH201812). References [1] W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J. He, Cancer statistics in China, 2015, CA Cancer J. Clin. 66 (2016) 115–132. [2] S. Lheureux, M. Braunstein, A.M. Oza, Epithelial ovarian cancer: evolution of management in the era of precision medicine, CA Cancer J. Clin. 69 (2019) 280–304. [3] A. Shang, W. Wang, C. Gu, C. Chen, B. Zeng, Y. Yang, P. Ji, J. Sun, J. Wu, W. Lu, Z. Sun, D. Li, Long non-coding RNA HOTTIP enhances IL-6 expression to potentiate immune escape of ovarian cancer cells by upregulating the expression of PD-L1 in neutrophils, J. Exp. Clin. Cancer Res. 38 (2019) 411. [4] L. Liu, J. Yi, X. Deng, J. Yuan, B. Zhou, Z. Lin, Z. Zeng, MYH9 overexpression correlates with clinicopathological parameters and poor prognosis of epithelial ovarian cancer, Oncol. Lett. 18 (2019) 1049–1056. [5] B.D. Adams, A.L. Kasinski, F.J. Slack, Aberrant regulation and function of MicroRNAs in Cancer, Curr. Biol. 24 (2014) 762–776. [6] L. Paladini, L. Fabris, G. Bottai, C. Raschioni, G.A. Calin, L. Santarpia, Targeting microRNAs as key modulators of tumor immune response, J. Exp. Clin. Cancer Res. 35 (2016) 103. [7] J. Bertoli, C. Cava, I. Castiglioni, MicroRNAs: New Biomarkers for Diagnosis, Prognosis, Therapy Prediction and Therapeutic Tools for Breast Cancer, Theranostics 5 (2015) 1122–1143. [8] E. Lages, H. Ipas, A. Guttin, H. Nesr, F. Berger, J.P. Issartel, MicroRNAs: molecular features and role in cancer, Front. Biosci. 17 (2012) 2508–2540. [9] E. Lee, K. Ito, Y. Zhao, E.E. Schadt, H.Y. Irie, J. Zhu, Inferred miRNA activity identifies miRNA-mediated regulatory networks underlying multiple cancers, Bioinformatics 32 (2016) 96–105. [10] K.K. To, C.W. Tong, M. Wu, W.C. Cho, MicroRNAs in the prognosis and therapy of colorectal cancer: from bench to bedside, World J. Gastroenterol. 24 (2018) 2949–2973. [11] D.L. Gianpiero, M. Garofalo, C.M. Croce, microRNAs in cancer, Annu. Rev. Pathol. 9 (2014) 287–314. [12] H. Lan, H. Lu, X. Wang, H. Jin, MicroRNAs as potential biomarkers in Cancer: opportunities and challenges, Biomed Res. Int. 2015 (2015) 125094. [13] M.V. Iorio, C.M. Croce, MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics, A comprehensive review, EMBO Mol. Med. 9 (2017) 852. [14] S.L. Romero-Cordoba, I. Salido-Guadarrama, M. Rodriguez-Dorantes, A. HidalgoMiranda, miRNA biogenesis: biological impact in the development of cancer, Cancer Biol. Ther. 15 (2014) 1444–1455. [15] T.A. Farazi, J.I. Hoell, P. Morozov, T. Tuschl, microRNAs in human Cancer, Adv. Exp. Med. Biol. 774 (2013) 1–20. [16] P. Hydbring, Y. Wang, A. Fassl, X. Li, V. Matia, T. Otto, Y.J. Choi, K.E. Sweeney, J.M. Suski, H. Yin, R.L. Bogorad, S. Goel, H. Yuzugullu, K.J. Kauffman, J. Yang, C. Jin, Y. Li, D. Floris, R. Swanson, K. Ng, E. Sicinska, L. Anders, J.J. Zhao, K. Polyak, D.G. Anderson, C. Li, P. Sicinski, Cell cycle-targeting MicroRNAs as therapeutic tools against refractory cancers, Cancer Cell 31 (2017) 576–590. [17] C.S. Mak, M.M. Yung, L.M. Hui, L.L. Leung, R. Liang, K. Chen, S.S. Liu, Y. Qin, T.H. Leung, K.F. Lee, K.K. Chan, H.Y. Ngan, D.W. Chan, MicroRNA-141 enhances anoikis resistance in metastatic progression of ovarian cancer through targeting KLF12/Sp1/survivin axis, Mol. Cancer 16 (2017) 11. [18] S. Zhang, Z. Lu, A.K. Unruh, C. Ivan, K.A. Baggerly, G.A. Calin, Z. Li, R.C. Bast, X.F. Le, Clinically relevant microRNAs in ovarian Cancer, Mol. Cancer Res. 13 (2015) 393–401. [19] K.R. Delfino, S.L. Rodriguez-Zas, Transcription Factor-MicroRNA-Target gene networks associated with ovarian Cancer survival and recurrence, PLoS One 8 (2013) e58608. [20] T. Zhu, W. Gao, X. Chen, Y. Zhang, M. Wu, P. Zhang, S. Wang, A pilot study of circulating MicroRNA-125b as a diagnostic and prognostic biomarker for epithelial ovarian Cancer, Int. J. Gynecol. Cancer 27 (2017) 3–10. [21] Y. Kinose, K. Sawada, K. Nakamura, T. Kimura, The role of microRNAs in ovarian

10

Biomedicine & Pharmacotherapy 125 (2020) 109865

L. Liu, et al.

[55] M. Zhao, P. Xu, Z. Liu, Y. Zhen, Y. Chen, Y. Liu, Q. Fu, X. Deng, Z. Liang, Y. Li, X. Lin, W. Fang, Dual roles of miR-374a by modulated c-Jun respectively targets CCND1-inducing PI3K/AKT signal and PTEN-suppressing Wnt/beta-catenin signaling in non-small-cell lung cancer, Cell Death Dis. 9 (2018) 78. [56] Q. Fu, X. Song, Z. Liu, X. Deng, R. Luo, C. Ge, R. Li, Z. Li, M. Zhao, Y. Chen, X. Lin, Q. Zhang, W. Fang, miRomics and proteomics reveal a miR-296-3p/PRKCA/FAK/ Ras/c-Myc feedback loop modulated by HDGF/DDX5/beta-catenin complex in lung adenocarcinoma, Clin. Cancer Res. 23 (2017) 336–6350. [57] Y. Zhen, W. Fang, M. Zhao, R. Luo, Y. Liu, Q. Fu, Y. Chen, C. Cheng, Y. Zhang, Z. Liu, miR-374a-CCND1-pPI3K/AKT-c-JUN feedback loop modulated by PDCD4 suppresses cell growth, metastasis, and sensitizes nasopharyngeal carcinoma to cisplatin, Oncogene 36 (2017) 275–285. [58] M. Zhao, R. Luo, Y. Liu, L. Gao, Z. Fu, Q. Fu, X. Luo, Y. Chen, X. Deng, Z. Liang, X. Li, C. Cheng, Z. Liu, W. Fang, miR-3188 regulates nasopharyngeal carcinoma proliferation and chemosensitivity through a FOXO1-modulated positive feedback loop with mTOR-p-PI3K/AKT-c-JUN, Nat. Commun. 7 (2016) 11309. [59] D. Cao, Y. Luo, S. Qin, M. Yu, Y. Mu, G. Ye, N. Yang, Z. Cong, J. Chen, J. Qin, R. Cui, R. Jing, H. Cao, M. Zhong, Metallopanstimulin-1 (MPS-1) mediates the promotion effect of leptin on colorectal cancer through activation of JNK/c-Jun signaling pathway, Cell Death Dis. 10 (2019) 655. [60] Z.Y. Lin, G. Chen, Y.Q. Zhang, H.C. He, Y.X. Liang, J.H. Ye, Y.K. Liang, R.J. Mo, J.M. Lu, Y.J. Zhuo, Y. Zheng, F.N. Jiang, Z.D. Han, S.L. Wu, W.D. Zhong, C.L. Wu, Correction to: MicroRNA-30d promotes angiogenesis and tumor growth via MYPT1/c-JUN/VEGFA pathway and predicts aggressive outcome in prostate cancer, Mol. Cancer 18 (2019) s12943. [61] X. Lin, S. Zuo, R. Luo, Y. Li, G. Yu, Y. Zou, Y. Zhou, Z. Liu, Y. Liu, Y. Hu, Y. Xie, W. Fang, Z. Liu, HBX-induced miR-5188 impairs FOXO1 to stimulate β-catenin nuclear translocation and promotes tumor stemness in hepatocellular carcinoma, Theranostics 9 (2019) 7583–7598.

[47] S.D. Coaxum, J. Tiedeken, E. Garrett-Mayer, J. Myers, S.A. Rosenzweig, D.M. Neskey, The tumor suppressor capability of p53 is dependent on non-muscle myosin IIA function in head and neck cancer, Oncotarget 8 (2017) 22991–23007. [48] M.A. Conti, A.D. Saleh, L.R. Brinster, H. Cheng, Z. Chen, S. Cornelius, C. Liu, X. Ma, W.C. Van, R.S. Adelstein, Conditional deletion of nonmuscle myosin II-A in mouse tongue epithelium results in squamous cell carcinoma, Sci. Rep. 5 (2015) 14068. [49] S.M. Kas, J.R. de Ruiter, K. Schipper, S. Annunziato, E. Schut, S. Klarenbeek, A.P. Drenth, E. Burg, C. Klijn, J.J. Ten Hoeve, D.J. Adams, M.J. Koudijs, J. Wesseling, M. Nethe, L.F.A. Wessels, J. Jonkers, Insertional mutagenesis identifies drivers of a novel oncogenic pathway in invasive lobular breast carcinoma, Nat. Genet. 49 (2017) 1219–1230. [50] Z.K. Xia, Y.C. Yuan, N. Yin, B.L. Yin, Z.P. Tan, Y.R. Hu, Nonmuscle myosin IIA is associated with poor prognosis of esophageal squamous cancer, Dis. Esophagus 25 (2012) 427–436. [51] K. Katono, Y. Sato, S.X. Jiang, M. Kobayashi, R. Nagashio, S. Ryuge, E. Fukuda, N. Goshima, Y. Satoh, M. Saegusa, N. Masuda, Prognostic significance of MYH9 expression in resected non-small cell lung cancer, PLoS One 10 (2015) e0121460. [52] L. Derycke, C. Stove, A.S. Vercoutter-Edouart, W.O. De, L. Dollé, N. Colpaert, H. Depypere, J.C. Michalski, M. Bracke, The role of non-muscle myosin IIA in aggregation and invasion of human MCF-7 breast cancer cells, Int. J. Dev. Biol. 55 (2011) 835–840. [53] Y. Zou, X. Lin, J. Bu, Z. Lin, Y. Chen, Y. Qiu, H. Mo, Y. Tang, W. Fang, Z. Wu, Timeless-stimulated miR-5188-FOXO1/β-Catenin-c-Jun feedback loop promotes stemness via ubiquitination of β-Catenin in breast Cancer, Mol. Ther. 19 (2019) S1525. [54] Z. Liang, Z. Liu, C. Cheng, H. Wang, X. Deng, J. Liu, C. Liu, Y. Li, W. Fang, VPS33B interacts with NESG1 to modulate EGFR/PI3K/AKT/c-Myc/P53/miR-133a-3p signaling and induce 5-fluorouracil sensitivity in nasopharyngeal carcinoma, Cell Death Dis. 10 (2019) 305.

11