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Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p
Journal Pre-proofs Research paper Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p Zheng Ji, Jufang Chi, He Sun, Ao Ru, Tingjuan Ni, Jie Zhang, Fengchun Jiang, Haitao Lv, Fang Peng, Hangyuan Guo, Yi Chen PII: DOI: Reference:
S0378-1119(19)30802-9 https://doi.org/10.1016/j.gene.2019.144143 GENE 144143
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
13 July 2019 22 September 2019 23 September 2019
Please cite this article as: Z. Ji, J. Chi, H. Sun, A. Ru, T. Ni, J. Zhang, F. Jiang, H. Lv, F. Peng, H. Guo, Y. Chen, Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene.2019.144143
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© 2019 Published by Elsevier B.V.
Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p Zheng Ji1,* (email: [email protected]) Jufang Chi1,* (email: [email protected]) He Sun2 (email: [email protected]) Ao Ru3 (email: [email protected]) Tingjuan Ni4 (email: [email protected]) Jie Zhang5 (email: [email protected]) Fengchun Jiang6 (email: [email protected]) Haitao Lv1 (email: [email protected]) Fang Peng1 (email: [email protected]) Hangyuan Guo1 (email: [email protected]) Yi Chen7,# (email: [email protected]) *Both authors equally contributed to this work. 1Department
of Cardiology, Shaoxing People's Hospital (Shaoxing Hospital,
Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. 2Department
of Clinical Laboratory Center, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. 3Department
of Ultrasonography, the First People′s Hospital of Huzhou，
Huzhou 313000, Zhejiang Province, China. 4Zhejiang
University School of Medicine, Zhejiang University, Hangzhou
310058, Zhejiang Province, China. 5The
First Clinical Medical College, Wenzhou Medical University, Wenzhou
325000, Zhejiang Province, China. 6Department
of Cardiology, Zhejiang Integrated Traditional and Western
Medicine Hospital, Hangzhou 310000, Zhejiang Province, China.
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7Department
of Pediatrics, Shaoxing People's Hospital (Shaoxing Hospital,
Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. # Corresponding
author: Yi Chen; email: [email protected]; telephone number: +86-575-88229052; address: Department of Pediatrics, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing city, Zhejiang Province, China; postal code: 312000.
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Abstract Atherosclerosis is a common cardiovascular disorder and is characterized by damage of endothelial cells, cell inflammation, hyper-proliferation of vascular smooth muscle cells and the accumulation of extracellular lipids and fibrous tissues. In this study, we firstly examined the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) in homocysteine (Hcy)-stimulated human aortic smooth muscle cells (HASMCs), and then looked into the potential molecular signaling axis of linc-ROR in regulating the proliferation and migration of HASMCs. Hcy promoted HASMC proliferation and up-regulated linc-ROR expression. Functional studies showed that linc-ROR exerted enhanced actions on the proliferation and migration of HASMCs. In addition, linc-ROR acted as a competing endogenous RNA for miR-195-5p and repressed the miR-195-5p expression in HASMCs. Linc-ROR was up-regulated the miR-195-3p was down-regulated in the plasma from CAD patients when compared to normal controls. Furthermore, fibroblast growth factor 2 (FGF2) was identified as a target of miR-195-5p and was negatively regulated by miR-195-5p in HASMCs. The rescue experiments revealed that linc-ROR-mediated HASMC proliferation and migration may be via regulating miR-195-5p/FGF2 axis. LincROR inhibition blocked the miR-195-5p/FGF2 signaling in Hcy-treated HASMCs, and this effect may also involve in the miR-195-5p/FGF2 axis. To summarize, the data of the present study identified the up-regulation of linc-ROR in Hcystimulated HASMCs, and further mechanistic functional studies revealed that linc-ROR promoted HASMC proliferation and migration via regulating miR-1955p/FGF2 axis. The present study provided the novel actions of linc-ROR in regulating HASMC proliferation and migration, which may be related to the pathophysiology of atherosclerosis. Keywords: atherosclerosis; linc-ROR; Hcy; HASMC; miR-195-5p; FGF2
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1. Introduction Atherosclerosis is a common cardiovascular disorder and is characterized by impairment of endothelial cells, cell inflammation, hyper-proliferation of vascular smooth muscle cells (VSMCs) and the accumulation of extracellular lipids and fibrous tissues (Abdolmaleki et al., 2018; Shah, 2019). Atherosclerosis may lead to serious clinical complications such as stroke, gangrene and myocardial infarction (Barton, 2013). Elevated levels of blood homocysteine (Hcy) are one of the important risk factors for the progression of atherosclerosis, where homocysteine acts to promote VSMC proliferation via different mechanisms (McCully, 2015; Sreckovic et al., 2017). Indeed, there is growing evidence showing that the activation as well as hyper-proliferative features of VSMCs under the pathological stimuli including Hcy play an important role in disease progression of atherosclerosis (Zhang et al., 2016; Ma et al., 2017; Ma et al., 2018). Therefore, restoring the dysfunction of VSMCs may provide effective approach to alleviate the progression of atherosclerosis. Long non-coding RNAs (lncRNAs) belong to a class of transcripts with more than 200 nucleotides and lack the capacity of coding proteins (Hung et al., 2018; Zhang et al., 2018). Up to date, various studies have illustrated the diverse functions of lncRNAs in regulating the biological processes such as cell proliferation and migration, cell apoptosis, cell differentiation and cell metabolism (Barman et al., 2019). In terms of VSMC functions, lncRNAs have been revealed for their essential roles in regulating VSMC proliferation, apoptosis and migration, which may eventually relate to the pathophysiology of atherosclerosis (Jian et al., 2016; Leung et al., 2016). Tan et al., found the dysregulation of lncRNA antisense non-coding RNA in the INK4 locus (ANRIL) in aging VSMCs and lncRNA ANRIL could enhance VSMC viability and induce VSMC senescence (Tan et al., 2019). LncRNA-H19 was identified to be associated with acute stroke treatment subtypes in atherosclerotic patients, and enforced H19 expression promoted VSMC proliferation and inhibited VSMC apoptosis, which may lead to the increased size of atherosclerotic plaque (Huang et al., 2019). Zhou et al., showed that lncRNA colorectal neoplasia differentially expressed regulated VSMC proliferation and migration under the pathological stimuli of plateletderived growth factor-bb (Zhou et al., 2019). Zhang et al., found that lncRNA maternally expressed 8 exerted inhibitory effects on VSMC proliferation and migration by targeting peroxisome proliferator-activated receptor alpha (Huo et al., 2019). Recently, the long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) has been identified as an oncogene in several types of cancers and promoted cancer cell proliferation and migration (Pan et al., 2016). However, as far as we known, the role of linc-ROR in regulating VSMC function has not been studied yet. 4
In this study, we firstly examined the expression level of linc-ROR in Hcystimulated human aortic smooth muscle cells (HASMCs), and then looked into the potential molecular signaling axis of linc-ROR in regulating the proliferation and migration of HASMCs. The present study may provide some preliminary insights into the role of linc-ROR in development and progression of atherosclerosis.
2. Materials and methods 2.1. Cell culture The HASMCs were purchased from Lonza (Basel, Switzerland) and the cells were cultured in high glucose (4.5 g/L) DMEM with the 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, USA) as the supplement. All the HASMCs were maintained in a humidified incubator with 5% CO2 at 37 oC.
2.2. Chemicals, oligonucleotides and cell transfections The homocysteine (Hcy; purity >95%) was purchased from Sigma-Aldrich (St. Louis, USA), and the final concentrations of Hcy in the culture medium for cell treatment were 50, 100, 200 and 500 µM, and the treatment duration was 6, 12 or 24 h. For the construction of the overexpression plasmids, pcDNA3.1 was used to generate the linc-ROR overexpressing (pcDNA3.1-linc-ROR) and fibroblast growth factor 2 (FGF2) overexpressing plasmids (pcDNA3.1-FGF2), and all the plasmids were from GenePharma (Shanghai, China). For the small interfering RNAs (siRNAs), the siRNAs for linc-ROR (si-linc-ROR#1 and si-lincROR#2) and the siRNA for FGF2 (si-FGF2) as well as the scrambled siRNAs (siNC) were all designed and synthesized by RiboBio (Guangzhou, China). The miRNA oligonucleotides including miR-195-5p mimics and its negative control (mimics ctrl), miR-195-5p inhibitors and its negative control (inhibitor ctrl) were purchased from QIAGEN (Hilden, Germany). The plasmids, siRNAs and miRNAs were transfected into HASMCs using Lipofectamine 3000 reagent (Thermo Fisher Scientific), and cells were collected from in vitro assays at 24 h after transfection.
2.3. Quantitative real-time PCR (qRT-PCR) Total RNA extraction from HASMCs were performed using TRIzol reagent (Invitrogen, Carlsbad, USA). The concentration of the extracted RNA was determined by measuring optical density at 260 nm. Total RNA was reversely transcribed into cDNA using Advantage RT-for-PCR kit (Clontech, Mountain View, 5
USA). The real-time PCR was performed in the StepOne Plus system (Applied Biosystems, Foster City, USA) using Fast SYBR Green Master Mix kit (Applied Biosystems). For the expression of linc-ROR, PCNA mRNA and FGF2 mRNA, GAPDH was used as the internal control, and for the expression of miR-195-5p, U6 was used as the internal control. The relative expression levels of the detected genes were calculated using the comparative cycle threshold method. The sequences for the primers were summarized in Supplementary Table S1.
2.4. Western blot assay Total cellular proteins from HASMCs were extracted using radioimmunoprecipitation assay buffer (Sigma-Aldrich), and equal amounts of extracted proteins were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis followed by electroblotting onto a polyvinylidene fluoride membrane using a semidry blotting method (Thermo Fisher Scientific). The membranes were blocked with 5% skimmed milk for 1 h at room temperature, and after blocking, the membranes were then incubated with corresponding primary antibodies against FGF2 (1:1000), phosphorylated phosphoinositide 3-kinase (p-PI3K; 1:1000), total PI3K (t-PI3K; 1:1000), phosphorylated AKT (p-AKT; 1:1500), total AKT (t-AKT; 1:1000) and β-actin (1:2000; Cell Signaling Technology, Danvers, USA) overnight at 4 oC. After overnight incubation, the membrane was washed with Tris-buffer saline with Tween 20 for 3 x 5 mins and were incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase for 2 h at room temperature. The protein bands were detected using the ECL kit (Thermo Fisher Scientific). The expression of β-actin protein was set as the loading control.
2.5. Cell Counting Kit-8 (CCK-8) assay The cell proliferation of HASMCs were assessed using the CCK-8 assay kit (Donjindo, Tokyo, Japan). Briefly, the HASMCs with different treatments were seeded in the 96-well-plates at a density of 1 x106 cells/well, after incubation for indicated durations, the HASMCs were incubated with the CCK-8 solution for 2 h at 37 oC. The HASMC proliferation was measured by determined the optical density of the re-acted solutions at a wavelength of 450 nm.
2.6. Transwell migration assay The HASMC migration was evaluated by the Transwell migration assay using the Transwell system (24-wells, 8 µm pore size with polycarbonate membrane; Corning Costar, Corning, USA). Briefly, HASMCs with different treatments were 6
seeded on the Transwell inserts at a density of 1 x105 cells/ well and the transwell inserts were filled with medium without serum, and the lower chamber was filled with medium with 10% FBS that serves as chemoattractant. The HASMCs were incubated for 24 h and the non-migrated cells on the top surface of the membranes was removed by cotton swabs, and the HASMCs migrated into the lower surface of the membrane were fixed with 100% methanol for 10 min and were then stained with 0.1% crystal violet for 10 min at room temperature. The number of migrated HASMCs were counted under a light microscope by randomly selecting 3 fields.
2.7. Luciferase reporter assay The luciferase reporter constructs that harbor the wild type fragments of lincROR and 3’ untranslated region (3’ UTR) of FGF2 were generated by cloning the PCR-amplified fragments into the pmiRGLO vector (Promega, Madison, USA). The corresponding mutant fragments were generated by the Site-Directed Mutagenesis kit (Stratagene, La Jolla, USA) and were subsequently cloned into the pmirGLO vector. For the luciferase reporter assay, the luciferase reporter constructs and miRNAs (mimics NC, miR-195-5p mimics, inhibitor NC and miR195-5p inhibitor) were co-transfected into HASMCs using Lipofectamine 3000 reagent (Invitrogen), and HASMCs were collected at 48 h after transfection for the determination of luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol.
2.9.
Clinical sample analysis
A total of 20 subjects, who underwent coronary angiography for known coronary atherosclerosis at the Shaoxing People's Hospital between 2016 and 2018 were enrolled in this study. Coronary artery disease (CAD) was diagnosed by angiography showing at least one segment of a major coronary artery (Zhang et al., 2017). A total of 20 subjects with normal coronary arteries were recruited as control groups. All the subjects had no family history of CAD and no history of significant concomitant diseases. Fasting blood samples were collected from each subject into a ethylenediamine tetra acetic acid tube, and plasma was collected and stored in -80 oC for further analysis. This study was approved by the Ethics Committee of Shaoxing People’s Hospital and written informed consent was obtained from all the recruited subjects.
2.10. Statistical analysis
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All the statistical analyses were performed using GraphPad Prism Version 6.0 (GraphPad Software, La Jolla, USA). All the data were presented as mean ± standard deviation. The significance of the statistical comparisons was tested by two-tailed Student’s t-test or one-way analysis of variance followed by Bonferroni’s post hoc test. A P value less than 0.05 was considered statistically significant.
3. Results 3.9. Hcy promoted HASMC proliferation and upregulated linc-ROR expression The effects of Hcy treatment (200 µM) for different durations on cell proliferation and linc-ROR expression were determined by CCK-8 and qRT-PCR assays, respectively, and Hcy time-dependently increased the HASMC proliferation and up-regulated linc-ROR expression in HASMCs (Fig. 1A and 1B). Furthermore, the effects of Hcy treatment for 24 h on cell proliferation of HASMCs were evaluated by CCK-8 assay, and expectedly, Hcy at concentrations from 50 to 500 µM increased the cell proliferative potential in a concentration-dependent manner (Fig.1C). In addition, the expression of linc-ROR was determined by qRT-PCR in HASMCs after being incubated with different concentrations of Hcy for 24 h, and Hcy concentration dependently up-regulated linc-ROR expression in HASMCs (Fig.1D). Hcy at 200 µM was found to exert the sub-maximal effects, and this concentration was selected in the subsequent studies.
3.10. Linc-ROR promoted HASMC proliferation and migration As linc-ROR expression was affected by Hcy treatment, the effects of linc-ROR on the HASMC proliferation and migration were assessed using gain-of- and loss-of-function assays. For the gain-of-function assay, the effects of linc-ROR overexpression on HASMC proliferation and migration were determined. As shown in Fig. 2A, linc-ROR overexpression was detected in HASMCs with pcDNA3.1-linc-ROR transient transfection when compared to pcDNA3.1 transfection (control group). Linc-ROR overexpression increased the cell proliferative index of HASMCs as determined by CCK-8 assay (Fig.2B), upregulated PCNA mRNA expression as determined by qRT-PCR (Fig.2C), and increased the number of migrated HASMCs were also significantly increased in the pcDNA3.1-linc-ROR transfection group as compared to pcDNA3.1 transfection group (Fig. 2D). For the loss-of-function assay, the effects of lincROR knockdown on HASMC proliferation and migration were evaluated. As shown in Fig.2E, linc-ROR knockdown was confirmed in HASMCs after being transiently transfected with linc-ROR siRNAs (linc-ROR#1 and #2). Knockdown 8
of linc-ROR significantly suppressed HASMC proliferation and migration (Supplementary Figure S1). Further CCK-8, qRT-PCR and Transwell migration assays showed that 200 µM treatment significantly increased the proliferative, PCNA mRNA expression level and migratory potentials of HASMCs as compared to control group (cells were cultured with normal culture medium; Fig.2F-2H); linc-ROR knockdown by the siRNA technique significantly suppressed the cell proliferation, PCNA mRNA expression level and migration of HASMCs with the treatment of 200 µM Hcy (Fig.2F-2H).
3.11. Linc-ROR sponged miR-195-5p and suppressed its expression in HASMCs As one of the important mechanistic actions of lncRNAs was acting as competing endogenous RNAs for miRNAs, the miRNAs that could be potentially sponged by linc-ROR was predicted by the LncBase Predicted V2.0 software. Among these predicted miRNAs, miR-195-5p was selected based on the literatures documenting the role of miR-195-5p in regulating cell proliferation and migration (Sun et al., 2017; Yang et al., 2019). The predicted binding sites between lincROR and miR-195-5p were shown in Fig.3A. The overexpression or knockdown of miR-195-5p were identified in HASMCs with miR-195-5p mimic or inhibitor transfection, respectively (Fig.3B). The effects of miR-195-5p on the luciferase activity of linc-ROR (WT) or (MUT) reporter vector were evaluated by DualLuciferase Reporter Assay, as shown in Fig.3C and 3D, miR-195-5p overexpression suppressed the luciferase activity of linc-ROR (WT) reporter vector and miR-195-5p knockdown increased the luciferase activity of linc-ROR (WT) reporter vector (Fig.3C); the mutation in the binding sites abolished the effects of miR-195-5p on the luciferase activity (Fig.3D). Expectedly, linc-ROR overexpression “sponged” miR-195-5p and repressed miR-195-5p expression in HASMCs (Fig. 3E), while linc-ROR silence caused an increase in miR-195-5p expression in HASMCs (Fig. 3F). In addition, 200 µM Hcy treatment for 24 h also significantly suppressed the expression of miR-195-5p in HASMCs (Fig. 3G). Furthermore, linc-ROR knockdown had a partial restoring effect on the miR-1955p in Hcy-treated HASMCs (Fig.3H). The examination of clinical samples showed that linc-ROR was up-regulated the miR-195-3p was down-regulated in the plasma from CAD patients when compared to normal controls (Fig. 3I and 3J).
3.12. MiR-195-5p targeted FGF2 and suppressed its expression in HASMCs As miRNA commonly binds to the 3’UTR of targeted genes to regulate gene expression, which subsequently affected the cellular functions, the targeted genes for miR-195-5p was evaluated by the online TargetScan tool. FGF2 was 9
among the predicted targets and was selected for examination due to its role in regulating VSMC proliferation and migration (Alhendi et al., 2018). The reporter vector containing WT or MUT 3’UTR of FGF2 based on the predicting results (Fig. 4A) was constructed using pGL3 vector. MiR-195-5p overexpression increased the luciferase activity of the WT reporter vector and miR-195-5p knockdown suppressed the luciferase activity of the WT reporter vector in HASMCs (Fig. 4B); on the other hand, the luciferase activity of MUT reporter vector was unaffected in HASMCs with different miRNAs transfection (Fig.4C). Moreover, the effects of miR-195-5p on the mRNA and protein expression of FGF2 was determined by qRT-PCR and western blot, and miR-195-5p overexpression down-regulated FGF2 expression, while miR-195-5p knockdown up-regulated FGF2 expression in HASMCs (Fig. 4D and 4E). Consistently, linc-ROR overexpression and 200 µM Hcy treatment for 24 h both significantly up-regulated the expression of FGF2 in HASMCs (Fig. 4F-4I). Moreover, linc-ROR knockdown had a partial restoring effect on the FGF2 expression level in Hcy-treated HASMCs (Fig. 4J and 4K).
3.13. Linc-ROR regulated HASMC proliferation and migration via miR-1955p/FGF2 axis The mechanisms of linc-ROR-mediated HASMC proliferation and migration were determine by the rescue experiments. The knockdown of FGF2 was confirmed in HASMCs transfected with FGF2 siRNA (si-FGF2), and with si-FGF2 transfection, the mRNA and protein expression of FGF2 was significantly repressed in HASMCs (Fig. 5A and 5B). The functional assays showed that the enforced miR195-5p overexpression and FGF2 knockdown both significantly attenuated the promoting effects of linc-ROR on the HASMC proliferation and migration (Fig. 5C and 5D). Transfection with pcDNA3.1-FGF2 caused a markedly increase in the mRNA and protein expression of FGF2 in HASMCs when compared to pcDNA3.1 transfection (Fig. 5E and 5F). Moreover, the miR-195-5p knockdown and enforced FGF2 overexpression attenuated the inhibitory effects of linc-ROR knockdown on the cell proliferation and migration in Hcy-treated HASMCs (Fig. 5G and 5H).
3.14. Linc-ROR regulated PI3K/AKT signaling pathways via targeting miR195-5p/FGF2 Linc-ROR was demonstrated to affect the PI3K/AKT signaling pathways in the cancer studies, and our data showed that overexpression of linc-ROR significantly increased the p-PI3K and p-AKT protein expression level, but not the protein expression levels of t-PI3K and t-AKT, and the enhanced actions of lincROR overexpression was partially prevented by the presence of miR-195-5p mimics or FGF2 siRNA in the HASMCs (Fig.6). 10
4. Discussion Homocysteine is generated during the process of sulfur amino acid metabolism, and studies have demonstrated that elevated levels of homocysteine in the peripheral blood were associated with the increased risk in the development of atherosclerosis (Temple et al., 2000). In vitro studies showed that homocysteine could promote the VSMC proliferation via enhancing DNA synthesis (Zou et al., 2010), suggesting the link between hyperproliferation of VSMCs induced by homocysteine and development of atherosclerosis. In the present study, we confirmed the enhanced effects of Hcy on the HASMC proliferation, and this effect was accompanied by the up-regulation of linc-ROR, suggesting that lincROR may participate in regulating the proliferative potentials of HASMCs.
Linc-ROR is a large intergenic ncRNA with ~2600 nucleotides in length and is first identified in induced pluripotent stem cells (Loewer et al., 2010). In the cancer studies, linc-ROR acts an oncogene to promote cancer cell proliferation and migration in various types of cancers. Linc-ROR could induce epithelial-tomesenchymal transition, which leads to the breast cancer tumorigenesis and metastasis (Hou et al., 2014). Linc-ROR can serve as a decoy oncoRNA that modifies the pattern of histone modification that promotes tumorigenesis (Fan et al., 2015). Linc-ROR also promoted gastric cancer stem cell proliferation, invasion and stemness (Wang et al., 2016). In the cardiovascular research, inhibition of linc-ROR was effective in suppressing the proliferation and migration of microvascular endothelial cells (Qin et al., 2018). Shen et al., revealed the potential role of linc-ROR for mediating the reprograming in the cardiac hypertrophy (Shen et al., 2017). In the in vitro functional studies, linc-ROR overexpression showed enhanced effects on HASMC proliferation and migration; while linc-ROR knockdown attenuated Hcy-mediated effects on HASMCs. Collectively, these data suggest that up-regulation of linc-ROR may be associated with hyper-proliferation and hyper-migration in VSMCs.
The downstream signaling pathway for linc-ROR was further determined firstly using the bioinformatics prediction, and miR-195-5p was selected for further examination in our study, as miR-195-5p was well-studied for its role in repressing tumor cell proliferation and migration in different types of cancers such as osteosarcoma, prostate cancer, colorectal cancer and breast cancer (Cai et al., 2018; Yang et al., 2018; Bai et al., 2019; Shen et al., 2019). In addition, miR-195-5p inhibited the angiogenesis in human prostate cancer via suppressing proline rich 11 expression (Cai et al., 2018). Evidence from pre-eclampsia studies showed that miR-195-5p exerted anti-angiogenic actions in endothelial 11
cells (Sandrim et al., 2016). Our data demonstrated the involvement of lincROR/miR-195-5p axis in regulating HASMC functions. Furthermore, the 3’UTRs that could be potentially targeted by miR-195-5p were predicted by TargetScan tool, and FGF2 was selected in the current investigations, as FGF2 has long been regarded as key mediators in the pathobiology of VSMCs. Previous studies have confirmed that FGF2 could promote VSMC proliferation and migration. In our study, we showed that FGF2 was negative regulated by miR-195-5p and was positively regulated by linc-ROR. More importantly, the rescue experiments showed that linc-ROR-mediated HASMC proliferation and migration may be via targeting miR-195-5p-FGF2 axis. Data from previous studies also showed that linc-ROR promoted lung cancer cell and endometrial cell proliferation and migration via PI3K/AKT signaling pathway (Shi et al., 2017; Xu et al., 2018). In this regard, we further explored potential interaction between linc-ROR and PI3K/AKT signaling in HASMCs. Our data showed that linc-ROR overexpression significantly activated the PI3K/AKT signaling, which was attenuated by miR-1955p mimics or FGF2 knockdown, suggesting the linc-ROR regulating PI3K/AKT signaling may be via miR-195-5p/FGF2 axis in HASMCs.
In the present study, we are aware of several limitations. The present study mainly performed in vitro assays, and further studies may select proper in vivo animal models to verify the current findings. The number of clinical sample was relatively small, and larger clinical sample size may be considered in the future studies. The signaling pathway in the present study was only evaluated in one cell line, and more cell lines may be examined to confirm the signaling pathway in controlling SMC proliferation and migration.
Collectively, the data of the present study identified the up-regulation of linc-ROR in Hcy-stimulated HASMCs, and further mechanistic functional studies revealed that linc-ROR promoted HASMC proliferation and migration via regulating miR195-5p/FGF2 axis. The present study provided the novel actions of linc-ROR in regulating VSMC proliferation and migration, which may be related to the pathophysiology of atherosclerosis.
Conflicts of interest None.
Acknowledgements 12
This project was supported by the General Research project of Science Technology Department of Zhejiang Province (2016C3322), the Research Fund of the Health Bureau of Zhejiang Province (2018KY172), the Young Scholar Fund of Shaoxing People’s Hospital (2018YB04) and the Joint Research project of the Health Bureau of Zhejiang Province (WKJ-ZJ-1729).
Abbreviations 3’ UTR, 3’ untranslated region; CCK-8, cell counting kit-8; FBS, fetal bovine serum; FGF2, fibroblast growth factor 2; HASMC, human aortic smooth muscle cell; Hcy, homocysteine; linc-ROR, long intergenic non-protein coding RNA, regulator of reprogramming; ;lncRNAs, long non-coding RNAs; MUT, mutant; qRT-PCR, quantitative real-time PCR; siRNAs, small interfering RNAs; VSMCs, vascular smooth muscle cells; WT, wild type.
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Hung, J., Miscianinov, V., Sluimer, J.C., Newby, D.E. and Baker, A.H., 2018. Targeting Non-coding RNA in Vascular Biology and Disease. Front Physiol 9, 1655. Huo, L., Wang, B., Zheng, M., Zhang, Y., Xu, J., Yang, G. and Guan, Q., 2019. miR-128-3p inhibits glioma cell proliferation and differentiation by targeting NPTX1 through IRS-1/PI3K/AKT signaling pathway. Exp Ther Med 17, 2921-2930. Jian, L., Jian, D., Chen, Q. and Zhang, L., 2016. Long Noncoding RNAs in Atherosclerosis. J Atheroscler Thromb 23, 376-84. Leung, A., Stapleton, K. and Natarajan, R., 2016. Functional Long Non-coding RNAs in Vascular Smooth Muscle Cells. Curr Top Microbiol Immunol 394, 127-41. Loewer, S., Cabili, M.N., Guttman, M., Loh, Y.H., Thomas, K., Park, I.H., Garber, M., Curran, M., Onder, T., Agarwal, S., Manos, P.D., Datta, S., Lander, E.S., Schlaeger, T.M., Daley, G.Q. and Rinn, J.L., 2010. Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells. Nat Genet 42, 1113-7. Ma, S.C., Cao, J.C., Zhang, H.P., Jiao, Y., Zhang, H., He, Y.Y., Wang, Y.H., Yang, X.L., Yang, A.N., Tian, J., Zhang, M.H., Yang, X.M., Lu, G.J., Jin, S.J., Jia, Y.X. and Jiang, Y.D., 2017. Aberrant promoter methylation of multiple genes in VSMC proliferation induced by Hcy. Mol Med Rep 16, 7775-7783. Ma, S.C., Zhang, H.P., Jiao, Y., Wang, Y.H., Zhang, H., Yang, X.L., Yang, A.N. and Jiang, Y.D., 2018. Homocysteine-induced proliferation of vascular smooth muscle cells occurs via PTEN hypermethylation and is mitigated by Resveratrol. Mol Med Rep 17, 5312-5319. McCully, K.S., 2015. Homocysteine and the pathogenesis of atherosclerosis. Expert Rev Clin Pharmacol 8, 211-9. Pan, Y., Li, C., Chen, J., Zhang, K., Chu, X., Wang, R. and Chen, L., 2016. The Emerging Roles of Long Noncoding RNA ROR (lincRNA-ROR) and its Possible Mechanisms in Human Cancers. Cell Physiol Biochem 40, 219-229. Qin, W.W., Xin, Z.L., Wang, H.Q., Wang, K.P., Li, X.Y. and Wang, X., 2018. Inhibiting lncRNA ROR suppresses growth, migration and angiogenesis in microvascular endothelial cells by upregulating miR-26. Eur Rev Med Pharmacol Sci 22, 7985-7993. Sandrim, V.C., Dias, M.C., Bovolato, A.L., Tanus-Santos, J.E., Deffune, E. and Cavalli, R.C., 2016. Plasma from pre-eclamptic patients induces the expression of the anti-angiogenic miR195-5p in endothelial cells. J Cell Mol Med 20, 1198-200. Shah, P.K., 2019. Inflammation, infection and atherosclerosis. Trends Cardiovasc Med. Shen, M., Dong, C., Ruan, X., Yan, W., Cao, M., Pizzo, D., Wu, X., Yang, L., Liu, L., Ren, X. and Wang, S.E., 2019. Chemotherapy-Induced Extracellular Vesicle miRNAs Promote Breast Cancer Stemness by Targeting ONECUT2. Cancer Res 79, 3608-3621. Shen, S., Jiang, H., Bei, Y., Xiao, J. and Li, X., 2017. Long Non-Coding RNAs in Cardiac Remodeling. Cell Physiol Biochem 41, 1830-1837. Shi, H., Pu, J., Zhou, X.L., Ning, Y.Y. and Bai, C., 2017. Silencing long non-coding RNA ROR improves sensitivity of non-small-cell lung cancer to cisplatin resistance by inhibiting PI3K/Akt/mTOR signaling pathway. Tumour Biol 39, 1010428317697568. Sreckovic, B., Sreckovic, V.D., Soldatovic, I., Colak, E., Sumarac-Dumanovic, M., Janeski, H., Janeski, N., Gacic, J. and Mrdovic, I., 2017. Homocysteine is a marker for metabolic syndrome and atherosclerosis. Diabetes Metab Syndr 11, 179-182. Sun, M., Song, H., Wang, S., Zhang, C., Zheng, L., Chen, F., Shi, D., Chen, Y., Yang, C., Xiang, Z., Liu, Q., Wei, C. and Xiong, B., 2017. Integrated analysis identifies microRNA-195 as a suppressor of Hippo-YAP pathway in colorectal cancer. J Hematol Oncol 10, 79.
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Tan, P., Guo, Y.H., Zhan, J.K., Long, L.M., Xu, M.L., Ye, L., Ma, X.Y., Cui, X.J. and Wang, H.Q., 2019. LncRNA-ANRIL inhibits cell senescence of vascular smooth muscle cells by regulating miR-181a/Sirt1. Biochem Cell Biol. Temple, M.E., Luzier, A.B. and Kazierad, D.J., 2000. Homocysteine as a risk factor for atherosclerosis. Ann Pharmacother 34, 57-65. Wang, S., Liu, F., Deng, J., Cai, X., Han, J. and Liu, Q., 2016. Long Noncoding RNA ROR Regulates Proliferation, Invasion, and Stemness of Gastric Cancer Stem Cell. Cell Reprogram 18, 319-326. Xu, X.Y., Zhang, J., Qi, Y.H., Kong, M., Liu, S.A. and Hu, J.J., 2018. Linc-ROR promotes endometrial cell proliferation by activating the PI3K-Akt pathway. Eur Rev Med Pharmacol Sci 22, 2218-2225. Yang, C., Wu, K., Wang, S. and Wei, G., 2018. Long non-coding RNA XIST promotes osteosarcoma progression by targeting YAP via miR-195-5p. J Cell Biochem 119, 5646-5656. Yang, R., Xing, L., Zheng, X., Sun, Y., Wang, X. and Chen, J., 2019. The circRNA circAGFG1 acts as a sponge of miR-195-5p to promote triple-negative breast cancer progression through regulating CCNE1 expression. Mol Cancer 18, 4. Zhang, H.P., Wang, Y.H., Cao, C.J., Yang, X.M., Ma, S.C., Han, X.B., Yang, X.L., Yang, A.N., Tian, J., Xu, H., Zhang, M.H. and Jiang, Y.D., 2016. A regulatory circuit involving miR-143 and DNMT3a mediates vascular smooth muscle cell proliferation induced by homocysteine. Mol Med Rep 13, 483-90. Zhang, Z., Gao, W., Long, Q.Q., Zhang, J., Li, Y.F., Liu, D.C., Yan, J.J., Yang, Z.J. and Wang, L.S., 2017. Increased plasma levels of lncRNA H19 and LIPCAR are associated with increased risk of coronary artery disease in a Chinese population. Sci Rep 7, 7491. Zhang, Z., Salisbury, D. and Sallam, T., 2018. Long Noncoding RNAs in Atherosclerosis: JACC Review Topic of the Week. J Am Coll Cardiol 72, 2380-2390. Zhou, Y., He, X., Liu, R., Qin, Y., Wang, S., Yao, X., Li, C. and Hu, Z., 2019. LncRNA CRNDE regulates the proliferation and migration of vascular smooth muscle cells. J Cell Physiol. Zou, T., Yang, W., Hou, Z. and Yang, J., 2010. Homocysteine enhances cell proliferation in vascular smooth muscle cells: role of p38 MAPK and p47phox. Acta Biochim Biophys Sin (Shanghai) 42, 908-15.
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Figure legends Figure 1. Hcy promoted HASMC proliferation and upregulated linc-ROR expression. The HASMCs were incubated with 200 µM for 0, 6, 12 and 24 h respectively, (A) the HASMC proliferation was evaluated by CCK-8 assay; (B) the expression of linc-ROR in HASMCs were determined by qRT-PCR. (C) The HASMCs were incubated with different concentrations of Hcy (50, 100, 200 and 500 µM) for 24 h, and the HASMC proliferation was evaluated by CCK-8 assay; (D) The expression of linc-ROR was determined by qRT-PCR in HASMCs with Hcy treatment for 24 h. N = 3. *P<0.05, **P<0.01 and ***P<0.001 versus the control group. Figure 2. Linc-ROR promoted HASMC proliferation and migration. For the gainof-function assays, the HASMCs were transiently transfected with pcDNA3.1 (Control group) and pcDNA3.1-linc-ROR (Linc-ROR group); and at 24 h after the in vitro transfections, (A) the expression of linc-ROR was determined by qRTPCR in HASMCs; (B) the HASMC proliferation was determined by CCK-8 assay; (C) the PCNA mRNA expression level was assessed by qRT-PCR; (D) the number of migrated HASMCs were measured by Trasnwell migration assay. For the loss-of-function assays, the HASMCs were transiently transfected with scrambled siRNA (si-NC group) or linc-ROR siRNAs (si-Lic-ROR#1 and #2 groups), and at 24 h after the in vitro transfections, (E) the expression of lincROR was determined by qRT-PCR in HASMCs; (F) the cell proliferation was determined by CCK-8 assay in Hcy-treated HASMCs; (G) the PCNA mRNA expression was assessed by qRT-PCR; (H) the migratory ability of Hcy-treated HASMCs was evaluated by Transwell migration assay. Control group was treated with normal culture medium. N = 3; significant differences between different treatment groups were indicated as *P<0.05, **P<0.01 and ***P<0.001. Figure 3. Linc-ROR sponged miR-195-5p and suppressed its expression in HASMCs. (A) The potential interacting sites between linc-ROR and miR-195-5p as predicted by LncBase Predicted V2.0. (B) The HASMCs were transiently transfected with different miRNAs, and 24 h later, the expression of miR-195-5p was measured by qRT-PCR. (C) The HASMCs were transiently co-transfected with reporter vector containing wild type (WT) linc-ROR fragments and respective miRNA oligonucleotides, and luciferase activity was assessed at 48 h after the co-transfection. (D) The HASMCs were transiently co-transfected with reporter vector containing mutant (MUT) linc-ROR fragments and respective miRNA oligonucleotides, and luciferase activity was assessed at 48 h after the cotransfection. (E) The expression of miR-195-5p was determined by qRT-PCR in HASMCs after in vitro transfection with pcDNA3.1 or pcDNA3.1-linc-ROR. (F) The expression of miR-195-5p was determined by qRT-PCR in HASMCs after treating with Hcy (200 µM) or control for 24 h. (G) The effects of linc-ROR knockdown on miR-195-5p expression were evaluated by qRT-PCR in Hcy16
treated HASMCs. (H) The expression of linc-ROR and (I) the expression of miR195-5p were determined by qRT-PCR in plasma from normal 20 controls and 20 CAD patients. N = 3, significant differences between different treatment groups were indicated as *P<0.05, **P<0.01 and ***P<0.001. Figure 4. MiR-195-5p targeted FGF2 and suppressed its expression in HASMCs. (A) The potential interacting sites between miR-195-5p and FGF2 3’UTR as predicted by TargetScan. (B) The HASMCs were transiently co-transfected with reporter vector containing wild type (WT) 3’UTR of FGF2 and respective miRNA oligonucleotides, and luciferase activity was assessed at 48 h after the cotransfection. (C) The HASMCs were transiently co-transfected with reporter vector containing mutant (MUT) 3’UTR of FGF2 and respective miRNA oligonucleotides, and luciferase activity was assessed at 48 h after the cotransfection. (D-E) The mRNA and protein expression of FGF2 in HASMCs after being transfected with different miRNAs were determined by qRT-PCR and western blot, respectively. (F-G) The mRNA and protein expression of FGF2 was determined by qRT-PCR and western blot in HASMCs after in vitro transfection with pcDNA3.1 or pcDNA3.1-linc-ROR. (H-I) (F) The mRNA and protein expression of FGF2 was determined by qRT-PCR and western blot in HASMCs after treating with Hcy (200 µM) or control for 24 h. (J-K) The effects of linc-ROR knockdown on FGF2 expression were evaluated by qRT-PCR and western blot in Hcy-treated HASMCs. N = 3, significant differences between different treatment groups were indicated as *P<0.05, **P<0.01 and ***P<0.001. Figure 5. Linc-ROR regulated HASMC proliferation and migration via miR-1955p/FGF2 axis. (A-B) The mRNA and protein expression of FGF2 in HASMCs after being transfected with pcDNA3.1 (Control group) or pcDNA3.1-FGF2 (FGF2 group) were determined by qRT-PCR and western blot. (C-D) The cell proliferation and migration of HASMCs after being con-transfected with pcDNA3.1-lincROR + miR-195-5p, pcDNA3.1-lin-ROR + si-FGF2 or their respective controls were determined by CCK-8 and Transwell migration assays. (E-F) The mRNA and protein expression of FGF2 in HASMCs after being transfected with scrambled siRNA (si-NC) or FGF2 siRNA (si-FGF2) were determined by qRT-PCR and western blot. (G-H) The cell proliferation and migration of HASMCs after being con-transfected with si-linc-ROR#1 + miR-1955p inhibitor, si-linc-ROR#1 + pcDNA3.1-FGF2 or their respective controls were determined by CCK-8 and Transwell migration assays. N = 3, significant differences between different treatment groups were indicated as *P<0.05, **P<0.01 and ***P<0.001. Figure 6. Linc-ROR regulated PI3K/AKT signaling pathways via targeting miR150-5p/FGF2. The protein expression of p-PI3K, t-PI3K, p-AKT and t-AKT HASMCs after being con-transfected with pcDNA3.1-linc-ROR + miR-195-5p mimics, pcDNA3.1-linc-ROR + si-FGF2 or their respective controls were 17
measured by western blot. N = 3, significant differences between different treatment groups were indicated as *P<0.05. Supplementary Figure S1. Effects of linc-ROR knockdown on the HAMSC proliferation and migration. For the loss-of-function assays, the HASMCs were transiently transfected scrambled siRNA (si-NC group) or linc-ROR siRNAs (siLic-ROR#1 and #2 groups); and at 24 h after the in vitro transfections, (A) the HASMC proliferation was determined by CCK-8 assay; (B) the PCNA mRNA expression level was assessed by qRT-PCR; (C) the number of migrated HASMCs were measured by Trasnwell migration assay.
Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p Zheng Ji1,* (email: [email protected]) Jufang Chi1,* (email: [email protected]) He Sun2 (email: [email protected]) Ao Ru3 (email: [email protected]) Tingjuan Ni4 (email: [email protected]) Jie Zhang5 (email: [email protected]) Fengchun Jiang6 (email: [email protected]) Haitao Lv1 (email: [email protected]) Fang Peng1 (email: [email protected]) Hangyuan Guo1 (email: [email protected]) Yi Chen7,# (email: [email protected]) *Both authors equally contributed to this work. 1Department
of Cardiology, Shaoxing People's Hospital (Shaoxing Hospital,
Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. 2Department
of Clinical Laboratory Center, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. 3Department
of Ultrasonography, the First People′s Hospital of Huzhou，
Huzhou 313000, Zhejiang Province, China. 18
4Zhejiang
University School of Medicine, Zhejiang University, Hangzhou
310058, Zhejiang Province, China. 5The
First Clinical Medical College, Wenzhou Medical University, Wenzhou
325000, Zhejiang Province, China. 6Department
of Cardiology, Zhejiang Integrated Traditional and Western
Medicine Hospital, Hangzhou 310000, Zhejiang Province, China. 7Department
of Pediatrics, Shaoxing People's Hospital (Shaoxing Hospital,
Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. # Corresponding
author: Yi Chen; email: [email protected]; telephone number: +86-575-88229052; address: Department of Pediatrics, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing city, Zhejiang Province, China; postal code: 312000.
Highlights
Homocysteine promoted HASMC proliferation and up-regulated linc-ROR expression. Linc-ROR promoted the proliferation and migration of HASMCs. Linc-ROR acted as a competing endogenous RNA for miR-195-5p. Linc-ROR-mediated HASMC proliferation and migration may be via regulating miR-195-5p/FGF2 axis.
Abbreviations 3’ UTR, 3’ untranslated region; CCK-8, cell counting kit-8; FBS, fetal bovine serum; FGF2, fibroblast growth factor 2; HASMC, human aortic smooth muscle cell; Hcy, homocysteine; linc-ROR, long intergenic non-protein coding RNA, regulator of reprogramming; ;lncRNAs, long non-coding RNAs; MUT, mutant; qRT-PCR, quantitative real-time PCR; siRNAs, small interfering RNAs; VSMCs, vascular smooth muscle cells; WT, wild type.
19
Conflict of interest None.
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
20
S0378-1119(19)30802-9 https://doi.org/10.1016/j.gene.2019.144143 GENE 144143
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
13 July 2019 22 September 2019 23 September 2019
Please cite this article as: Z. Ji, J. Chi, H. Sun, A. Ru, T. Ni, J. Zhang, F. Jiang, H. Lv, F. Peng, H. Guo, Y. Chen, Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene.2019.144143
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Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p Zheng Ji1,* (email: [email protected]) Jufang Chi1,* (email: [email protected]) He Sun2 (email: [email protected]) Ao Ru3 (email: [email protected]) Tingjuan Ni4 (email: [email protected]) Jie Zhang5 (email: [email protected]) Fengchun Jiang6 (email: [email protected]) Haitao Lv1 (email: [email protected]) Fang Peng1 (email: [email protected]) Hangyuan Guo1 (email: [email protected]) Yi Chen7,# (email: [email protected]) *Both authors equally contributed to this work. 1Department
of Cardiology, Shaoxing People's Hospital (Shaoxing Hospital,
Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. 2Department
of Clinical Laboratory Center, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. 3Department
of Ultrasonography, the First People′s Hospital of Huzhou，
Huzhou 313000, Zhejiang Province, China. 4Zhejiang
University School of Medicine, Zhejiang University, Hangzhou
310058, Zhejiang Province, China. 5The
First Clinical Medical College, Wenzhou Medical University, Wenzhou
325000, Zhejiang Province, China. 6Department
of Cardiology, Zhejiang Integrated Traditional and Western
Medicine Hospital, Hangzhou 310000, Zhejiang Province, China.
1
7Department
of Pediatrics, Shaoxing People's Hospital (Shaoxing Hospital,
Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. # Corresponding
author: Yi Chen; email: [email protected]; telephone number: +86-575-88229052; address: Department of Pediatrics, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing city, Zhejiang Province, China; postal code: 312000.
2
Abstract Atherosclerosis is a common cardiovascular disorder and is characterized by damage of endothelial cells, cell inflammation, hyper-proliferation of vascular smooth muscle cells and the accumulation of extracellular lipids and fibrous tissues. In this study, we firstly examined the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) in homocysteine (Hcy)-stimulated human aortic smooth muscle cells (HASMCs), and then looked into the potential molecular signaling axis of linc-ROR in regulating the proliferation and migration of HASMCs. Hcy promoted HASMC proliferation and up-regulated linc-ROR expression. Functional studies showed that linc-ROR exerted enhanced actions on the proliferation and migration of HASMCs. In addition, linc-ROR acted as a competing endogenous RNA for miR-195-5p and repressed the miR-195-5p expression in HASMCs. Linc-ROR was up-regulated the miR-195-3p was down-regulated in the plasma from CAD patients when compared to normal controls. Furthermore, fibroblast growth factor 2 (FGF2) was identified as a target of miR-195-5p and was negatively regulated by miR-195-5p in HASMCs. The rescue experiments revealed that linc-ROR-mediated HASMC proliferation and migration may be via regulating miR-195-5p/FGF2 axis. LincROR inhibition blocked the miR-195-5p/FGF2 signaling in Hcy-treated HASMCs, and this effect may also involve in the miR-195-5p/FGF2 axis. To summarize, the data of the present study identified the up-regulation of linc-ROR in Hcystimulated HASMCs, and further mechanistic functional studies revealed that linc-ROR promoted HASMC proliferation and migration via regulating miR-1955p/FGF2 axis. The present study provided the novel actions of linc-ROR in regulating HASMC proliferation and migration, which may be related to the pathophysiology of atherosclerosis. Keywords: atherosclerosis; linc-ROR; Hcy; HASMC; miR-195-5p; FGF2
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1. Introduction Atherosclerosis is a common cardiovascular disorder and is characterized by impairment of endothelial cells, cell inflammation, hyper-proliferation of vascular smooth muscle cells (VSMCs) and the accumulation of extracellular lipids and fibrous tissues (Abdolmaleki et al., 2018; Shah, 2019). Atherosclerosis may lead to serious clinical complications such as stroke, gangrene and myocardial infarction (Barton, 2013). Elevated levels of blood homocysteine (Hcy) are one of the important risk factors for the progression of atherosclerosis, where homocysteine acts to promote VSMC proliferation via different mechanisms (McCully, 2015; Sreckovic et al., 2017). Indeed, there is growing evidence showing that the activation as well as hyper-proliferative features of VSMCs under the pathological stimuli including Hcy play an important role in disease progression of atherosclerosis (Zhang et al., 2016; Ma et al., 2017; Ma et al., 2018). Therefore, restoring the dysfunction of VSMCs may provide effective approach to alleviate the progression of atherosclerosis. Long non-coding RNAs (lncRNAs) belong to a class of transcripts with more than 200 nucleotides and lack the capacity of coding proteins (Hung et al., 2018; Zhang et al., 2018). Up to date, various studies have illustrated the diverse functions of lncRNAs in regulating the biological processes such as cell proliferation and migration, cell apoptosis, cell differentiation and cell metabolism (Barman et al., 2019). In terms of VSMC functions, lncRNAs have been revealed for their essential roles in regulating VSMC proliferation, apoptosis and migration, which may eventually relate to the pathophysiology of atherosclerosis (Jian et al., 2016; Leung et al., 2016). Tan et al., found the dysregulation of lncRNA antisense non-coding RNA in the INK4 locus (ANRIL) in aging VSMCs and lncRNA ANRIL could enhance VSMC viability and induce VSMC senescence (Tan et al., 2019). LncRNA-H19 was identified to be associated with acute stroke treatment subtypes in atherosclerotic patients, and enforced H19 expression promoted VSMC proliferation and inhibited VSMC apoptosis, which may lead to the increased size of atherosclerotic plaque (Huang et al., 2019). Zhou et al., showed that lncRNA colorectal neoplasia differentially expressed regulated VSMC proliferation and migration under the pathological stimuli of plateletderived growth factor-bb (Zhou et al., 2019). Zhang et al., found that lncRNA maternally expressed 8 exerted inhibitory effects on VSMC proliferation and migration by targeting peroxisome proliferator-activated receptor alpha (Huo et al., 2019). Recently, the long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) has been identified as an oncogene in several types of cancers and promoted cancer cell proliferation and migration (Pan et al., 2016). However, as far as we known, the role of linc-ROR in regulating VSMC function has not been studied yet. 4
In this study, we firstly examined the expression level of linc-ROR in Hcystimulated human aortic smooth muscle cells (HASMCs), and then looked into the potential molecular signaling axis of linc-ROR in regulating the proliferation and migration of HASMCs. The present study may provide some preliminary insights into the role of linc-ROR in development and progression of atherosclerosis.
2. Materials and methods 2.1. Cell culture The HASMCs were purchased from Lonza (Basel, Switzerland) and the cells were cultured in high glucose (4.5 g/L) DMEM with the 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, USA) as the supplement. All the HASMCs were maintained in a humidified incubator with 5% CO2 at 37 oC.
2.2. Chemicals, oligonucleotides and cell transfections The homocysteine (Hcy; purity >95%) was purchased from Sigma-Aldrich (St. Louis, USA), and the final concentrations of Hcy in the culture medium for cell treatment were 50, 100, 200 and 500 µM, and the treatment duration was 6, 12 or 24 h. For the construction of the overexpression plasmids, pcDNA3.1 was used to generate the linc-ROR overexpressing (pcDNA3.1-linc-ROR) and fibroblast growth factor 2 (FGF2) overexpressing plasmids (pcDNA3.1-FGF2), and all the plasmids were from GenePharma (Shanghai, China). For the small interfering RNAs (siRNAs), the siRNAs for linc-ROR (si-linc-ROR#1 and si-lincROR#2) and the siRNA for FGF2 (si-FGF2) as well as the scrambled siRNAs (siNC) were all designed and synthesized by RiboBio (Guangzhou, China). The miRNA oligonucleotides including miR-195-5p mimics and its negative control (mimics ctrl), miR-195-5p inhibitors and its negative control (inhibitor ctrl) were purchased from QIAGEN (Hilden, Germany). The plasmids, siRNAs and miRNAs were transfected into HASMCs using Lipofectamine 3000 reagent (Thermo Fisher Scientific), and cells were collected from in vitro assays at 24 h after transfection.
2.3. Quantitative real-time PCR (qRT-PCR) Total RNA extraction from HASMCs were performed using TRIzol reagent (Invitrogen, Carlsbad, USA). The concentration of the extracted RNA was determined by measuring optical density at 260 nm. Total RNA was reversely transcribed into cDNA using Advantage RT-for-PCR kit (Clontech, Mountain View, 5
USA). The real-time PCR was performed in the StepOne Plus system (Applied Biosystems, Foster City, USA) using Fast SYBR Green Master Mix kit (Applied Biosystems). For the expression of linc-ROR, PCNA mRNA and FGF2 mRNA, GAPDH was used as the internal control, and for the expression of miR-195-5p, U6 was used as the internal control. The relative expression levels of the detected genes were calculated using the comparative cycle threshold method. The sequences for the primers were summarized in Supplementary Table S1.
2.4. Western blot assay Total cellular proteins from HASMCs were extracted using radioimmunoprecipitation assay buffer (Sigma-Aldrich), and equal amounts of extracted proteins were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis followed by electroblotting onto a polyvinylidene fluoride membrane using a semidry blotting method (Thermo Fisher Scientific). The membranes were blocked with 5% skimmed milk for 1 h at room temperature, and after blocking, the membranes were then incubated with corresponding primary antibodies against FGF2 (1:1000), phosphorylated phosphoinositide 3-kinase (p-PI3K; 1:1000), total PI3K (t-PI3K; 1:1000), phosphorylated AKT (p-AKT; 1:1500), total AKT (t-AKT; 1:1000) and β-actin (1:2000; Cell Signaling Technology, Danvers, USA) overnight at 4 oC. After overnight incubation, the membrane was washed with Tris-buffer saline with Tween 20 for 3 x 5 mins and were incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase for 2 h at room temperature. The protein bands were detected using the ECL kit (Thermo Fisher Scientific). The expression of β-actin protein was set as the loading control.
2.5. Cell Counting Kit-8 (CCK-8) assay The cell proliferation of HASMCs were assessed using the CCK-8 assay kit (Donjindo, Tokyo, Japan). Briefly, the HASMCs with different treatments were seeded in the 96-well-plates at a density of 1 x106 cells/well, after incubation for indicated durations, the HASMCs were incubated with the CCK-8 solution for 2 h at 37 oC. The HASMC proliferation was measured by determined the optical density of the re-acted solutions at a wavelength of 450 nm.
2.6. Transwell migration assay The HASMC migration was evaluated by the Transwell migration assay using the Transwell system (24-wells, 8 µm pore size with polycarbonate membrane; Corning Costar, Corning, USA). Briefly, HASMCs with different treatments were 6
seeded on the Transwell inserts at a density of 1 x105 cells/ well and the transwell inserts were filled with medium without serum, and the lower chamber was filled with medium with 10% FBS that serves as chemoattractant. The HASMCs were incubated for 24 h and the non-migrated cells on the top surface of the membranes was removed by cotton swabs, and the HASMCs migrated into the lower surface of the membrane were fixed with 100% methanol for 10 min and were then stained with 0.1% crystal violet for 10 min at room temperature. The number of migrated HASMCs were counted under a light microscope by randomly selecting 3 fields.
2.7. Luciferase reporter assay The luciferase reporter constructs that harbor the wild type fragments of lincROR and 3’ untranslated region (3’ UTR) of FGF2 were generated by cloning the PCR-amplified fragments into the pmiRGLO vector (Promega, Madison, USA). The corresponding mutant fragments were generated by the Site-Directed Mutagenesis kit (Stratagene, La Jolla, USA) and were subsequently cloned into the pmirGLO vector. For the luciferase reporter assay, the luciferase reporter constructs and miRNAs (mimics NC, miR-195-5p mimics, inhibitor NC and miR195-5p inhibitor) were co-transfected into HASMCs using Lipofectamine 3000 reagent (Invitrogen), and HASMCs were collected at 48 h after transfection for the determination of luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol.
2.9.
Clinical sample analysis
A total of 20 subjects, who underwent coronary angiography for known coronary atherosclerosis at the Shaoxing People's Hospital between 2016 and 2018 were enrolled in this study. Coronary artery disease (CAD) was diagnosed by angiography showing at least one segment of a major coronary artery (Zhang et al., 2017). A total of 20 subjects with normal coronary arteries were recruited as control groups. All the subjects had no family history of CAD and no history of significant concomitant diseases. Fasting blood samples were collected from each subject into a ethylenediamine tetra acetic acid tube, and plasma was collected and stored in -80 oC for further analysis. This study was approved by the Ethics Committee of Shaoxing People’s Hospital and written informed consent was obtained from all the recruited subjects.
2.10. Statistical analysis
7
All the statistical analyses were performed using GraphPad Prism Version 6.0 (GraphPad Software, La Jolla, USA). All the data were presented as mean ± standard deviation. The significance of the statistical comparisons was tested by two-tailed Student’s t-test or one-way analysis of variance followed by Bonferroni’s post hoc test. A P value less than 0.05 was considered statistically significant.
3. Results 3.9. Hcy promoted HASMC proliferation and upregulated linc-ROR expression The effects of Hcy treatment (200 µM) for different durations on cell proliferation and linc-ROR expression were determined by CCK-8 and qRT-PCR assays, respectively, and Hcy time-dependently increased the HASMC proliferation and up-regulated linc-ROR expression in HASMCs (Fig. 1A and 1B). Furthermore, the effects of Hcy treatment for 24 h on cell proliferation of HASMCs were evaluated by CCK-8 assay, and expectedly, Hcy at concentrations from 50 to 500 µM increased the cell proliferative potential in a concentration-dependent manner (Fig.1C). In addition, the expression of linc-ROR was determined by qRT-PCR in HASMCs after being incubated with different concentrations of Hcy for 24 h, and Hcy concentration dependently up-regulated linc-ROR expression in HASMCs (Fig.1D). Hcy at 200 µM was found to exert the sub-maximal effects, and this concentration was selected in the subsequent studies.
3.10. Linc-ROR promoted HASMC proliferation and migration As linc-ROR expression was affected by Hcy treatment, the effects of linc-ROR on the HASMC proliferation and migration were assessed using gain-of- and loss-of-function assays. For the gain-of-function assay, the effects of linc-ROR overexpression on HASMC proliferation and migration were determined. As shown in Fig. 2A, linc-ROR overexpression was detected in HASMCs with pcDNA3.1-linc-ROR transient transfection when compared to pcDNA3.1 transfection (control group). Linc-ROR overexpression increased the cell proliferative index of HASMCs as determined by CCK-8 assay (Fig.2B), upregulated PCNA mRNA expression as determined by qRT-PCR (Fig.2C), and increased the number of migrated HASMCs were also significantly increased in the pcDNA3.1-linc-ROR transfection group as compared to pcDNA3.1 transfection group (Fig. 2D). For the loss-of-function assay, the effects of lincROR knockdown on HASMC proliferation and migration were evaluated. As shown in Fig.2E, linc-ROR knockdown was confirmed in HASMCs after being transiently transfected with linc-ROR siRNAs (linc-ROR#1 and #2). Knockdown 8
of linc-ROR significantly suppressed HASMC proliferation and migration (Supplementary Figure S1). Further CCK-8, qRT-PCR and Transwell migration assays showed that 200 µM treatment significantly increased the proliferative, PCNA mRNA expression level and migratory potentials of HASMCs as compared to control group (cells were cultured with normal culture medium; Fig.2F-2H); linc-ROR knockdown by the siRNA technique significantly suppressed the cell proliferation, PCNA mRNA expression level and migration of HASMCs with the treatment of 200 µM Hcy (Fig.2F-2H).
3.11. Linc-ROR sponged miR-195-5p and suppressed its expression in HASMCs As one of the important mechanistic actions of lncRNAs was acting as competing endogenous RNAs for miRNAs, the miRNAs that could be potentially sponged by linc-ROR was predicted by the LncBase Predicted V2.0 software. Among these predicted miRNAs, miR-195-5p was selected based on the literatures documenting the role of miR-195-5p in regulating cell proliferation and migration (Sun et al., 2017; Yang et al., 2019). The predicted binding sites between lincROR and miR-195-5p were shown in Fig.3A. The overexpression or knockdown of miR-195-5p were identified in HASMCs with miR-195-5p mimic or inhibitor transfection, respectively (Fig.3B). The effects of miR-195-5p on the luciferase activity of linc-ROR (WT) or (MUT) reporter vector were evaluated by DualLuciferase Reporter Assay, as shown in Fig.3C and 3D, miR-195-5p overexpression suppressed the luciferase activity of linc-ROR (WT) reporter vector and miR-195-5p knockdown increased the luciferase activity of linc-ROR (WT) reporter vector (Fig.3C); the mutation in the binding sites abolished the effects of miR-195-5p on the luciferase activity (Fig.3D). Expectedly, linc-ROR overexpression “sponged” miR-195-5p and repressed miR-195-5p expression in HASMCs (Fig. 3E), while linc-ROR silence caused an increase in miR-195-5p expression in HASMCs (Fig. 3F). In addition, 200 µM Hcy treatment for 24 h also significantly suppressed the expression of miR-195-5p in HASMCs (Fig. 3G). Furthermore, linc-ROR knockdown had a partial restoring effect on the miR-1955p in Hcy-treated HASMCs (Fig.3H). The examination of clinical samples showed that linc-ROR was up-regulated the miR-195-3p was down-regulated in the plasma from CAD patients when compared to normal controls (Fig. 3I and 3J).
3.12. MiR-195-5p targeted FGF2 and suppressed its expression in HASMCs As miRNA commonly binds to the 3’UTR of targeted genes to regulate gene expression, which subsequently affected the cellular functions, the targeted genes for miR-195-5p was evaluated by the online TargetScan tool. FGF2 was 9
among the predicted targets and was selected for examination due to its role in regulating VSMC proliferation and migration (Alhendi et al., 2018). The reporter vector containing WT or MUT 3’UTR of FGF2 based on the predicting results (Fig. 4A) was constructed using pGL3 vector. MiR-195-5p overexpression increased the luciferase activity of the WT reporter vector and miR-195-5p knockdown suppressed the luciferase activity of the WT reporter vector in HASMCs (Fig. 4B); on the other hand, the luciferase activity of MUT reporter vector was unaffected in HASMCs with different miRNAs transfection (Fig.4C). Moreover, the effects of miR-195-5p on the mRNA and protein expression of FGF2 was determined by qRT-PCR and western blot, and miR-195-5p overexpression down-regulated FGF2 expression, while miR-195-5p knockdown up-regulated FGF2 expression in HASMCs (Fig. 4D and 4E). Consistently, linc-ROR overexpression and 200 µM Hcy treatment for 24 h both significantly up-regulated the expression of FGF2 in HASMCs (Fig. 4F-4I). Moreover, linc-ROR knockdown had a partial restoring effect on the FGF2 expression level in Hcy-treated HASMCs (Fig. 4J and 4K).
3.13. Linc-ROR regulated HASMC proliferation and migration via miR-1955p/FGF2 axis The mechanisms of linc-ROR-mediated HASMC proliferation and migration were determine by the rescue experiments. The knockdown of FGF2 was confirmed in HASMCs transfected with FGF2 siRNA (si-FGF2), and with si-FGF2 transfection, the mRNA and protein expression of FGF2 was significantly repressed in HASMCs (Fig. 5A and 5B). The functional assays showed that the enforced miR195-5p overexpression and FGF2 knockdown both significantly attenuated the promoting effects of linc-ROR on the HASMC proliferation and migration (Fig. 5C and 5D). Transfection with pcDNA3.1-FGF2 caused a markedly increase in the mRNA and protein expression of FGF2 in HASMCs when compared to pcDNA3.1 transfection (Fig. 5E and 5F). Moreover, the miR-195-5p knockdown and enforced FGF2 overexpression attenuated the inhibitory effects of linc-ROR knockdown on the cell proliferation and migration in Hcy-treated HASMCs (Fig. 5G and 5H).
3.14. Linc-ROR regulated PI3K/AKT signaling pathways via targeting miR195-5p/FGF2 Linc-ROR was demonstrated to affect the PI3K/AKT signaling pathways in the cancer studies, and our data showed that overexpression of linc-ROR significantly increased the p-PI3K and p-AKT protein expression level, but not the protein expression levels of t-PI3K and t-AKT, and the enhanced actions of lincROR overexpression was partially prevented by the presence of miR-195-5p mimics or FGF2 siRNA in the HASMCs (Fig.6). 10
4. Discussion Homocysteine is generated during the process of sulfur amino acid metabolism, and studies have demonstrated that elevated levels of homocysteine in the peripheral blood were associated with the increased risk in the development of atherosclerosis (Temple et al., 2000). In vitro studies showed that homocysteine could promote the VSMC proliferation via enhancing DNA synthesis (Zou et al., 2010), suggesting the link between hyperproliferation of VSMCs induced by homocysteine and development of atherosclerosis. In the present study, we confirmed the enhanced effects of Hcy on the HASMC proliferation, and this effect was accompanied by the up-regulation of linc-ROR, suggesting that lincROR may participate in regulating the proliferative potentials of HASMCs.
Linc-ROR is a large intergenic ncRNA with ~2600 nucleotides in length and is first identified in induced pluripotent stem cells (Loewer et al., 2010). In the cancer studies, linc-ROR acts an oncogene to promote cancer cell proliferation and migration in various types of cancers. Linc-ROR could induce epithelial-tomesenchymal transition, which leads to the breast cancer tumorigenesis and metastasis (Hou et al., 2014). Linc-ROR can serve as a decoy oncoRNA that modifies the pattern of histone modification that promotes tumorigenesis (Fan et al., 2015). Linc-ROR also promoted gastric cancer stem cell proliferation, invasion and stemness (Wang et al., 2016). In the cardiovascular research, inhibition of linc-ROR was effective in suppressing the proliferation and migration of microvascular endothelial cells (Qin et al., 2018). Shen et al., revealed the potential role of linc-ROR for mediating the reprograming in the cardiac hypertrophy (Shen et al., 2017). In the in vitro functional studies, linc-ROR overexpression showed enhanced effects on HASMC proliferation and migration; while linc-ROR knockdown attenuated Hcy-mediated effects on HASMCs. Collectively, these data suggest that up-regulation of linc-ROR may be associated with hyper-proliferation and hyper-migration in VSMCs.
The downstream signaling pathway for linc-ROR was further determined firstly using the bioinformatics prediction, and miR-195-5p was selected for further examination in our study, as miR-195-5p was well-studied for its role in repressing tumor cell proliferation and migration in different types of cancers such as osteosarcoma, prostate cancer, colorectal cancer and breast cancer (Cai et al., 2018; Yang et al., 2018; Bai et al., 2019; Shen et al., 2019). In addition, miR-195-5p inhibited the angiogenesis in human prostate cancer via suppressing proline rich 11 expression (Cai et al., 2018). Evidence from pre-eclampsia studies showed that miR-195-5p exerted anti-angiogenic actions in endothelial 11
cells (Sandrim et al., 2016). Our data demonstrated the involvement of lincROR/miR-195-5p axis in regulating HASMC functions. Furthermore, the 3’UTRs that could be potentially targeted by miR-195-5p were predicted by TargetScan tool, and FGF2 was selected in the current investigations, as FGF2 has long been regarded as key mediators in the pathobiology of VSMCs. Previous studies have confirmed that FGF2 could promote VSMC proliferation and migration. In our study, we showed that FGF2 was negative regulated by miR-195-5p and was positively regulated by linc-ROR. More importantly, the rescue experiments showed that linc-ROR-mediated HASMC proliferation and migration may be via targeting miR-195-5p-FGF2 axis. Data from previous studies also showed that linc-ROR promoted lung cancer cell and endometrial cell proliferation and migration via PI3K/AKT signaling pathway (Shi et al., 2017; Xu et al., 2018). In this regard, we further explored potential interaction between linc-ROR and PI3K/AKT signaling in HASMCs. Our data showed that linc-ROR overexpression significantly activated the PI3K/AKT signaling, which was attenuated by miR-1955p mimics or FGF2 knockdown, suggesting the linc-ROR regulating PI3K/AKT signaling may be via miR-195-5p/FGF2 axis in HASMCs.
In the present study, we are aware of several limitations. The present study mainly performed in vitro assays, and further studies may select proper in vivo animal models to verify the current findings. The number of clinical sample was relatively small, and larger clinical sample size may be considered in the future studies. The signaling pathway in the present study was only evaluated in one cell line, and more cell lines may be examined to confirm the signaling pathway in controlling SMC proliferation and migration.
Collectively, the data of the present study identified the up-regulation of linc-ROR in Hcy-stimulated HASMCs, and further mechanistic functional studies revealed that linc-ROR promoted HASMC proliferation and migration via regulating miR195-5p/FGF2 axis. The present study provided the novel actions of linc-ROR in regulating VSMC proliferation and migration, which may be related to the pathophysiology of atherosclerosis.
Conflicts of interest None.
Acknowledgements 12
This project was supported by the General Research project of Science Technology Department of Zhejiang Province (2016C3322), the Research Fund of the Health Bureau of Zhejiang Province (2018KY172), the Young Scholar Fund of Shaoxing People’s Hospital (2018YB04) and the Joint Research project of the Health Bureau of Zhejiang Province (WKJ-ZJ-1729).
Abbreviations 3’ UTR, 3’ untranslated region; CCK-8, cell counting kit-8; FBS, fetal bovine serum; FGF2, fibroblast growth factor 2; HASMC, human aortic smooth muscle cell; Hcy, homocysteine; linc-ROR, long intergenic non-protein coding RNA, regulator of reprogramming; ;lncRNAs, long non-coding RNAs; MUT, mutant; qRT-PCR, quantitative real-time PCR; siRNAs, small interfering RNAs; VSMCs, vascular smooth muscle cells; WT, wild type.
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Figure legends Figure 1. Hcy promoted HASMC proliferation and upregulated linc-ROR expression. The HASMCs were incubated with 200 µM for 0, 6, 12 and 24 h respectively, (A) the HASMC proliferation was evaluated by CCK-8 assay; (B) the expression of linc-ROR in HASMCs were determined by qRT-PCR. (C) The HASMCs were incubated with different concentrations of Hcy (50, 100, 200 and 500 µM) for 24 h, and the HASMC proliferation was evaluated by CCK-8 assay; (D) The expression of linc-ROR was determined by qRT-PCR in HASMCs with Hcy treatment for 24 h. N = 3. *P<0.05, **P<0.01 and ***P<0.001 versus the control group. Figure 2. Linc-ROR promoted HASMC proliferation and migration. For the gainof-function assays, the HASMCs were transiently transfected with pcDNA3.1 (Control group) and pcDNA3.1-linc-ROR (Linc-ROR group); and at 24 h after the in vitro transfections, (A) the expression of linc-ROR was determined by qRTPCR in HASMCs; (B) the HASMC proliferation was determined by CCK-8 assay; (C) the PCNA mRNA expression level was assessed by qRT-PCR; (D) the number of migrated HASMCs were measured by Trasnwell migration assay. For the loss-of-function assays, the HASMCs were transiently transfected with scrambled siRNA (si-NC group) or linc-ROR siRNAs (si-Lic-ROR#1 and #2 groups), and at 24 h after the in vitro transfections, (E) the expression of lincROR was determined by qRT-PCR in HASMCs; (F) the cell proliferation was determined by CCK-8 assay in Hcy-treated HASMCs; (G) the PCNA mRNA expression was assessed by qRT-PCR; (H) the migratory ability of Hcy-treated HASMCs was evaluated by Transwell migration assay. Control group was treated with normal culture medium. N = 3; significant differences between different treatment groups were indicated as *P<0.05, **P<0.01 and ***P<0.001. Figure 3. Linc-ROR sponged miR-195-5p and suppressed its expression in HASMCs. (A) The potential interacting sites between linc-ROR and miR-195-5p as predicted by LncBase Predicted V2.0. (B) The HASMCs were transiently transfected with different miRNAs, and 24 h later, the expression of miR-195-5p was measured by qRT-PCR. (C) The HASMCs were transiently co-transfected with reporter vector containing wild type (WT) linc-ROR fragments and respective miRNA oligonucleotides, and luciferase activity was assessed at 48 h after the co-transfection. (D) The HASMCs were transiently co-transfected with reporter vector containing mutant (MUT) linc-ROR fragments and respective miRNA oligonucleotides, and luciferase activity was assessed at 48 h after the cotransfection. (E) The expression of miR-195-5p was determined by qRT-PCR in HASMCs after in vitro transfection with pcDNA3.1 or pcDNA3.1-linc-ROR. (F) The expression of miR-195-5p was determined by qRT-PCR in HASMCs after treating with Hcy (200 µM) or control for 24 h. (G) The effects of linc-ROR knockdown on miR-195-5p expression were evaluated by qRT-PCR in Hcy16
treated HASMCs. (H) The expression of linc-ROR and (I) the expression of miR195-5p were determined by qRT-PCR in plasma from normal 20 controls and 20 CAD patients. N = 3, significant differences between different treatment groups were indicated as *P<0.05, **P<0.01 and ***P<0.001. Figure 4. MiR-195-5p targeted FGF2 and suppressed its expression in HASMCs. (A) The potential interacting sites between miR-195-5p and FGF2 3’UTR as predicted by TargetScan. (B) The HASMCs were transiently co-transfected with reporter vector containing wild type (WT) 3’UTR of FGF2 and respective miRNA oligonucleotides, and luciferase activity was assessed at 48 h after the cotransfection. (C) The HASMCs were transiently co-transfected with reporter vector containing mutant (MUT) 3’UTR of FGF2 and respective miRNA oligonucleotides, and luciferase activity was assessed at 48 h after the cotransfection. (D-E) The mRNA and protein expression of FGF2 in HASMCs after being transfected with different miRNAs were determined by qRT-PCR and western blot, respectively. (F-G) The mRNA and protein expression of FGF2 was determined by qRT-PCR and western blot in HASMCs after in vitro transfection with pcDNA3.1 or pcDNA3.1-linc-ROR. (H-I) (F) The mRNA and protein expression of FGF2 was determined by qRT-PCR and western blot in HASMCs after treating with Hcy (200 µM) or control for 24 h. (J-K) The effects of linc-ROR knockdown on FGF2 expression were evaluated by qRT-PCR and western blot in Hcy-treated HASMCs. N = 3, significant differences between different treatment groups were indicated as *P<0.05, **P<0.01 and ***P<0.001. Figure 5. Linc-ROR regulated HASMC proliferation and migration via miR-1955p/FGF2 axis. (A-B) The mRNA and protein expression of FGF2 in HASMCs after being transfected with pcDNA3.1 (Control group) or pcDNA3.1-FGF2 (FGF2 group) were determined by qRT-PCR and western blot. (C-D) The cell proliferation and migration of HASMCs after being con-transfected with pcDNA3.1-lincROR + miR-195-5p, pcDNA3.1-lin-ROR + si-FGF2 or their respective controls were determined by CCK-8 and Transwell migration assays. (E-F) The mRNA and protein expression of FGF2 in HASMCs after being transfected with scrambled siRNA (si-NC) or FGF2 siRNA (si-FGF2) were determined by qRT-PCR and western blot. (G-H) The cell proliferation and migration of HASMCs after being con-transfected with si-linc-ROR#1 + miR-1955p inhibitor, si-linc-ROR#1 + pcDNA3.1-FGF2 or their respective controls were determined by CCK-8 and Transwell migration assays. N = 3, significant differences between different treatment groups were indicated as *P<0.05, **P<0.01 and ***P<0.001. Figure 6. Linc-ROR regulated PI3K/AKT signaling pathways via targeting miR150-5p/FGF2. The protein expression of p-PI3K, t-PI3K, p-AKT and t-AKT HASMCs after being con-transfected with pcDNA3.1-linc-ROR + miR-195-5p mimics, pcDNA3.1-linc-ROR + si-FGF2 or their respective controls were 17
measured by western blot. N = 3, significant differences between different treatment groups were indicated as *P<0.05. Supplementary Figure S1. Effects of linc-ROR knockdown on the HAMSC proliferation and migration. For the loss-of-function assays, the HASMCs were transiently transfected scrambled siRNA (si-NC group) or linc-ROR siRNAs (siLic-ROR#1 and #2 groups); and at 24 h after the in vitro transfections, (A) the HASMC proliferation was determined by CCK-8 assay; (B) the PCNA mRNA expression level was assessed by qRT-PCR; (C) the number of migrated HASMCs were measured by Trasnwell migration assay.
Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p Zheng Ji1,* (email: [email protected]) Jufang Chi1,* (email: [email protected]) He Sun2 (email: [email protected]) Ao Ru3 (email: [email protected]) Tingjuan Ni4 (email: [email protected]) Jie Zhang5 (email: [email protected]) Fengchun Jiang6 (email: [email protected]) Haitao Lv1 (email: [email protected]) Fang Peng1 (email: [email protected]) Hangyuan Guo1 (email: [email protected]) Yi Chen7,# (email: [email protected]) *Both authors equally contributed to this work. 1Department
of Cardiology, Shaoxing People's Hospital (Shaoxing Hospital,
Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. 2Department
of Clinical Laboratory Center, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. 3Department
of Ultrasonography, the First People′s Hospital of Huzhou，
Huzhou 313000, Zhejiang Province, China. 18
4Zhejiang
University School of Medicine, Zhejiang University, Hangzhou
310058, Zhejiang Province, China. 5The
First Clinical Medical College, Wenzhou Medical University, Wenzhou
325000, Zhejiang Province, China. 6Department
of Cardiology, Zhejiang Integrated Traditional and Western
Medicine Hospital, Hangzhou 310000, Zhejiang Province, China. 7Department
of Pediatrics, Shaoxing People's Hospital (Shaoxing Hospital,
Zhejiang University School of Medicine), Shaoxing 312000, Zhejiang Province, China. # Corresponding
author: Yi Chen; email: [email protected]; telephone number: +86-575-88229052; address: Department of Pediatrics, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing city, Zhejiang Province, China; postal code: 312000.
Highlights
Homocysteine promoted HASMC proliferation and up-regulated linc-ROR expression. Linc-ROR promoted the proliferation and migration of HASMCs. Linc-ROR acted as a competing endogenous RNA for miR-195-5p. Linc-ROR-mediated HASMC proliferation and migration may be via regulating miR-195-5p/FGF2 axis.
Abbreviations 3’ UTR, 3’ untranslated region; CCK-8, cell counting kit-8; FBS, fetal bovine serum; FGF2, fibroblast growth factor 2; HASMC, human aortic smooth muscle cell; Hcy, homocysteine; linc-ROR, long intergenic non-protein coding RNA, regulator of reprogramming; ;lncRNAs, long non-coding RNAs; MUT, mutant; qRT-PCR, quantitative real-time PCR; siRNAs, small interfering RNAs; VSMCs, vascular smooth muscle cells; WT, wild type.
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Conflict of interest None.
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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