Cellular Signalling 26 (2014) 724–729
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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
MiR-21 inhibits c-Ski signaling to promote the proliferation of rat vascular smooth muscle cells Jun Li a,⁎, Li Zhao b, Xie He b, Ting Yang b, Kang Yang a a b
Department of Cardiothoracic Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing 400038, China
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Article history: Received 5 December 2013 Accepted 18 December 2013 Available online 31 December 2013 Keywords: miR-21 c-Ski VSMC Proliferation
a b s t r a c t Previously, we reported that the decrease of endogenous c-Ski expression is implicated in the progression of vascular smooth muscle cell (VSMC) proliferation after arterial injury. However, the molecular mechanism of the down-regulation of c-Ski is not clear. In this study, a potential miR-21 recognition element was identified in the 3′-untranslated region (UTR) of rat c-Ski mRNA. A reporter assay revealed that miR-21 could recognize the miR-21 recognition element of c-Ski mRNA. In A10 rat aortic smooth muscle cells, overexpression of miR-21 significantly inhibited the expression of c-Ski protein and promoted cell proliferation, which could be blocked by inhibition of miR-21 or overexpression of c-Ski. Further investigation demonstrated that the effect of miR-21 on VSMC proliferation resulted from negative regulation of c-Ski to suppress p38–p21/p27 signaling, the downstream pathway of c-Ski in VSMCs. These results indicate that c-Ski is a target gene of miR-21. miR-21 specifically binds to the 3′-untranslated region of c-Ski and negatively regulates c-Ski expression to diminish the protective effects of c-Ski and stimulate VSMC proliferation in the progression of arterial injury. © 2014 Elsevier Inc. All rights reserved.
1. Introduction The proliferation of vascular smooth muscle cells (VSMCs) following vascular injury is critical to the pathogenesis of intimal hyperplasia and atherosclerosis [1–3]. More studies are focusing on looking into the endogenous factors that either stimulate VSMCs to proliferate or try to protect against proliferation. For example, some findings demonstrated that decreased endogenous production of hydrogen sulfide accelerates atherosclerosis, endogenous estrogen deficiency reduces proliferation and enhances apoptosis-related death in vascular smooth muscle cells and endogenous parathyroid hormone-related protein regulates the expression of PTH type 1 receptor and proliferation of vascular smooth muscle cells [4–6]. Recently, we have reported that c-Ski, a molecule expressed in VSMCs, suppresses VSMC stimulation and intimal hyperplasia in a rat balloon injury model [7]. However, the expression of endogenous cSki in the arterial wall and VSMCs was found to decrease in a timedependent manner after vascular injury. These findings indicate that down-regulation of endogenous c-Ski expression is implicated in the Abbreviations: VSMC, vascular smooth muscle cell; UTR, untranslated region; miRNAs, microRNAs; FBS, fetal bovine serum; AMI, acute myocardial infarction; PDCD4, programmed cell death 4; PDCD4, activator protein 1; HSP70, heat-shock protein 70; TGFβ, transforming growth factor β; Shh, Sonic hedgehog; PTEN, phosphatase and tensin homology deleted from chromosome 10. ⁎ Corresponding author at: Department of Cardiothoracic Surgery, Southwest Hospital, Third Military Medical University, 30 Gaotanyan, Shapingba, Chongqing 400038, China. Tel./fax: +86 23 68765330. E-mail address: [email protected] (J. Li). 0898-6568/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.12.013
progression of VSMC proliferation after arterial injury. These findings prompted us to further investigate the mechanism of c-Ski regulation. MicroRNAs (miRNAs) are a subset of small non-coding RNAs, averaging 22 nucleotides in length [8]. In mammals, the main function of mature miRNAs is thought to be recognizing the 3-untranslated regions of specific target mRNAs to suppress translation and, occasionally, to induce mRNA degradation [9,10]. Therefore, miRNAs are considered to be important negative regulatory factors. Growing evidence indicates that miRNAs control the processes of development and differentiation in many organisms, and miRNA dysregulation has been correlated with the aggressiveness and progression of several diseases [11–13]. Additionally, multiple miRNAs are found to be involved in VSMC proliferation and atherosclerosis by regulating gene expression [14–16]. Accordingly, in this study, we investigated the effect and mechanism of miRNAs in the regulation of c-Ski expression in VSMCs. We identified a potential miR-21 recognition element in the 3′-untranslated region (UTR) of rat c-Ski mRNA and confirmed that miR-21 could recognize the element and negatively regulate c-Ski protein expression. In addition, miR-21 stimulated VSMC proliferation via c-Ski inhibition. This effect mainly resulted from suppression of the c-Ski–p38–p21/p27 signaling pathway.
2. Materials and methods All animal procedures were carried out in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals Guidelines by the Third Military Medical University on the
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ethical use of animals and were approved by the State Science and Technology Commission of China. 2.1. Balloon injury model As previously described [7,17,18], male Sprague–Dawley rats (450–500 g) were anesthetized, and the left common carotid artery was exposed for a small arteriotomy. A 2 F Fogarty balloon catheter (Edwards Lifescience, U.S.A.) was introduced, and the balloon, inflated by a 1.6-atm pressure, was inserted and withdrawn three times. 2.2. Microarray and bioinformatics analysis At 28 days after balloon injury, the normal and injured vascular tissues were collected. MiRNA expression profiling was determined by miRNA microarray analysis using rat miRNA array probes. The bioinformatics analysis for potential miRNAs that may regulate c-Ski was performed online using database searches (miRBase, TargetScan, miRanda). 2.3. Cell culture Rat aortic A10 VSMCs were obtained from the American Tissue Culture Collection and grown, as recommended, in modified DMEM containing 4 mmol/L L-glutamine, 4.5 g/L glucose, 1 mmol/L sodium pyruvate, and 1.5 g/L sodium bicarbonate, supplemented with 10% fetal bovine serum (FBS) and antibiotics. 2.4. Western blot On days 0, 7, 14 and 28 post-injury, c-Ski expression in the rat carotid artery was detected by western blot following standard procedure. To detect the effect of miR-21 on c-Ski expression and downstream signaling, A10 cells were treated with 50 nM miR-21 mimic or inhibitor or were previously transfected with Adc-Ski to overexpress c-Ski, as previously described [7]. Twelve hours after treatment, western blot analysis was performed to determine protein expression using antibodies against c-Ski (sc-33693, Santa Cruz), p-p38 (sc-365846, Santa Cruz), p21 (sc397, Santa Cruz) and p27 (sc 528; Santa Cruz). In these experiments, β-actin (sc-1616; Santa Cruz) served as the endogenous control. 2.5. Real-time RT-PCR for mature microRNA For the quantification of mature miR-21, polyadenylation and reverse transcription were performed using the All-in-One TM miRNA qRT-PCR Detection Kit (GeneCopoeia) according to the manufacturer's protocol. Quantitative PCR was then carried out with SYBR green detection, with U6 used as an internal control for template normalization. The primers for mature miR-21 and U6 were both provided by GeneCopoeia, Inc. 2.6. Luciferase assays The rat c-Ski mRNA fragment from 2218 to 2786 (No. XM233731) containing the miR-21 recognition element was commercially synthesized at Sunbio Medical Biotech (Shanghai, China) and cloned into the pMIR-REPORT vector (named pMIR-c-Ski) after digestion with XhoI and HindIII. The primers were 5′-CTGGAGAAGGTGGTGAAGGA-3′ (forward) and 5′-CACGATGCGAGGAGTG ATGT-3′ (reverse). The pMIR-cSki-mutant plasmid, where the miR-21 recognition element was mutated to “GCTGATCG,” was generated using the QuikChange site-directed mutagenesis kit (Stratagene, CA). Subsequently, A10 cells were transiently transfected using Lipofectamine2000 with luciferase reporter pMIR-REPORT, pMIR-c-Ski or pMIR-c-Ski mutant. The reporter constructs were transiently transfected with 50 nM miR-21 mimic, 50 nM miR-21 inhibitor or negative control. Transfection efficiency was
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monitored by co-transfection of pMIR-REPORT-β-gal. Cell extracts were prepared after transfection, and luciferase and β-galactosidase assays were performed according to the manufacturer's instruction. All transfection data were expressed as the ratios of luciferase activities/ β-galactosidase activities. 2.7. Cell proliferation assay Proliferation rates of A10 cells were determined by the Cell Count Kit-8 (CCK-8 Kit, Beyotime Inst Biotech, China) using the WST-8 (2(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2H-tetrazolium (monosodium salt)) dye method according to the manufacturer's instructions and references [7]. 2.8. Statistical analysis The values are expressed as the means ± SEM. An unpaired Student's t-test was used to evaluate the significant differences between the control and treated groups. Values of P b 0.05 were considered to be significant. All experiments were repeated at least three times. 3. Results 3.1. Endogenous c-Ski protein expression, rather than mRNA level, decreases in a time-dependent manner after vascular injury In the rat balloon injury model, endogenous c-Ski protein and mRNA levels were detected. As shown in Fig. 1A, no significant changes in c-Ski mRNA expression levels were observed during the 28 days postvascular injury. However, western blot results showed that 7 days after vascular injury, c-Ski protein expression decreased compared with the uninjured group. On days 14 and 28 post-injury, the decreases in c-Ski protein expression were significant (Fig. 1B). These data demonstrate that endogenous c-Ski protein, rather than mRNA, expression in the injured rat carotid arteries was significantly down-regulated in a time-dependent manner. These results indicate that c-Ski expression may be post-transcriptionally regulated. 3.2. miR-21 shows potential to regulate c-Ski via microarray and database searches To investigate whether c-Ski is modulated by miRNA, an important mechanism of post-transcriptional regulation, we assayed significantly changed miRNAs after vascular injury by microarray and investigated what potential miRNAs regulate c-Ski via online database searches (miRBase, TargetScan, miRanda). Compared with levels in normal control vessels, 8 miRNAs were found to have more than a five-fold increase in expression levels in balloon-injured arteries. However, among the aberrantly expressed miRNAs, only miR-21 was indicated to have the potential to regulate c-Ski by bioinformatics analysis (Fig. 2A). The potential miR-21 recognition element is located on +2516 to +2523 in the 3′-UTR of rat c-Ski mRNA (Fig. 2B). 3.3. Endogenous miR-21 levels in the arterial wall increase in a timedependent manner after vascular injury Using the rat balloon injury model, we then assayed the endogenous miR-21 expression levels on days 0, 7, 14 and 28 after vascular injury. Contrary to c-Ski protein expression levels, endogenous miR-21 levels in the arterial wall increased in a time-dependent manner during the 28 days following vascular injury (Fig. 3). These data further indicate that miR-21 may be involved in the down-regulation of c-Ski protein expression after vascular injury.
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Fig. 1. Time course of endogenous c-Ski expression after vascular injury. The c-Ski mRNA and protein expression levels in injured rat carotid arteries were detected on days 0, 7, 14 and 28 post-balloon angioplasty in rats. (A) RT-PCR for c-Ski mRNA expression in injured rat carotid arteries. (B) Western blot for c-Ski protein expression in injured rat carotid arteries. The representative results are from 3 separate experiments, and the data from RT-PCR experiments are expressed as the mean ± S.E.M.
3.4. miR-21 regulates c-Ski protein expression in A10 cells The effects of miR-21 on c-Ski protein levels in A10 cells were examined by western blot. A10 cells were transfected with an miR-21 mimic, a synthetic RNA duplex that mimics endogenous miR-21, which significantly increased miR-21 levels (Fig. 4A). This transfection induced a repression of c-Ski protein levels compared with the control (Fig. 4C). Conversely, transfection of the miR-21 inhibitor, a single-stranded modified RNA with a complimentary sequence to the mature miRNA that functions by sequestering/degrading endogenous miRNAs, markedly decreased miR-21 expression in A10 cells while subsequently significantly inducing c-Ski protein levels (Fig. 4B and C). 3.5. Validation of the miR-21 target site in the 3′-UTR of c-Ski mRNA Next, using a luciferase reporter assay, we tested whether miR-21 regulated c-Ski in A10 cells through direct targeting of the c-Ski 3′UTR. As shown in Fig. 5, the luciferase activity of pMIR-c-Ski plasmid, in which the miR-21 recognition element was inserted downstream of the luciferase gene, was down-regulated to some extent in the presence of the NC of the miR-21 mimic or inhibitor compared with the control (pMIR-REPORT). Because miR-21 is highly expressed in VSMCs, this may result from the effect of endogenous miR-21 on the luciferase activity of pMIR-c-Ski plasmid. It should be noted that, compared with the treatments of the NCs, the miR-21 mimic significantly decreased the luciferase activity of pMIR-c-Ski (Fig. 5A), while the miR-21 inhibitor restored the repression of luciferase activity of pMIR-c-Ski induced by endogenous miR-21 (Fig. 5B). However, these effects of the miR-21 mimic and inhibitor on the luciferase activity of the pMIR-c-Ski mutant
Fig. 2. Potential miRNA to regulate rat c-Ski expression. On day 28 after balloon injury, miRNAs with greater than a five-fold increase in expression were detected by miRNA microarray. Potential miRNAs to regulate c-Ski were identified by online database searches. (A) The intersection of miRNA microarray data and bioinformatics analysis online. (B) The potential binding site for miR-21 in the 3′-UTR of rat c-Ski mRNA.
plasmid (in which the miR-21 recognition element was mutated) were not observed (Fig. 5A and B). These data determine that miR-21 binds to the putative miR-21 target site within the c-Ski 3′-UTR in A10 cells. 3.6. miR-21 promotes the proliferation of A10 cells via regulation of c-Ski Our previous study reported that c-Ski plays a role in inhibiting VSMC proliferation [7]. Accordingly, to explore whether the miR-21mediated regulation of c-Ski affects the growth of VSMCs, the proliferation of A10 cells was investigated. As shown in Fig. 6, compared with the control, the miR-21 mimic stimulated, while the miR-21 inhibitor suppressed, A10 cell proliferation significantly. However, in A10 cells overexpressing c-Ski, the effect of the miR-21 mimic on the induction of cell proliferation was largely blocked, while the inhibitory role of the miR21 inhibitor was enhanced. These results suggest that miR-21 promotes the proliferation of A10 cells via regulation of c-Ski. 3.7. miR-21 modulation of c-Ski affects the expression of p38, p21 and p27 Because p38 and p21/p27 expression was demonstrated to be downstream of c-Ski and involved in mediating the inhibitory effect of c-Ski on VSMC proliferation, we then assayed the effect of miR-21 on p38–p21/p27 signaling. Western blot results showed that the miR-21 mimic suppressed, while the miR-21 inhibitor enhanced, p38 phosphorylation (Fig. 7A) and p21/p27 expression levels (Fig. 7B and C). The effects of the miR-21 mimic could be blocked both by the miR-21 inhibitor and the overexpression of c-Ski (Fig. 7). However, the NCs of the miR-21 mimic and miR-21 inhibitor had no effect on these molecules. These data confirm that miR-21 targets c-Ski to modulate the p38– p21/p27 signaling involved in the effect of miR-21 on VSMC proliferation, as described above.
Fig. 3. Time-course of endogenous miR-21 expression after vascular injury. The expression levels of miR-21 in injured rat carotid arteries were detected by RT-PCR on days 0, 7, 14 and 28 post-balloon angioplasty. The representative results are from 3 separate experiments, and the data are expressed as the mean ± S.E.M. * Indicates P b 0.01 compared to day 0.
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Fig. 4. Effects of overexpression or inhibition of miR-21 on c-Ski protein levels in A10 cells. The miR-21 mimic (50 nM), inhibitor (50 nM) or negative control was transfected into A10 cells. After 48 h, the cells were harvested and total RNA and protein extracts were isolated. (A) and (B) Levels of mature miR-21 expression were determined by RT-PCR. The values shown represent the mature miR-21 levels normalized to U6 snRNA levels, relative to controls. (C) c-Ski protein expression was determined by western blot. The representative results are from 3 separate experiments, and the data are expressed as the mean ± S.E.M. * Indicates P b 0.01 compared to the control.
4. Discussion c-Ski was originally discovered as the cellular homolog of retroviral v-Ski and was thus classified as a proto-oncogene [19,20]. In addition
to this, numerous recent studies have found more and more important functions of c-Ski in physiological and pathological conditions, such as in normal development and wound healing [21–23]. We previously reported that c-Ski has an inhibitory effect on VSMC proliferation and
Fig. 5. Validation of miR-421 target sites via luciferase reporter system. Reporter constructs were transiently transfected into A10 cells with 50 nM miR-21 mimic, inhibitor or their negative controls (NC), respectively. The relative luciferase activities were calculated as luciferase activities/β-galactosidase activities. (A) Luciferase activities in the presence of the miR-21 mimic. (B) Luciferase activities in the presence of the miR-21 inhibitor. The representative results are from 3 separate experiments, and the data are expressed as the mean ± S.E.M. * Indicates P b 0.01 when comparing the two groups.
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Fig. 6. Effect of miR-21 on A10 cell proliferation. The proliferation of A10 cells transfected with the miR-21 mimic or inhibitor was assayed using a CCK-8 Kit with the WST-8 dye method at 0 h, 24 h, 48 h and 72 h after transfection. In addition, to detect whether regulation of c-Ski was involved in the effect of miR-21, c-Ski was overexpressed in rat A10 cells by Adc-Ski transfection, as previously described. The representative results are from 3 separate experiments, and the data are expressed as the mean ± S.E.M. * Indicates P b 0.01 compared with the control; # Indicates P b 0.01 when comparing the two groups.
intimal hyperplasia in a rat balloon injury model. We also found that endogenous c-Ski expression in the arterial wall decreased in a timedependent manner after vascular injury [7]. In this study, we further showed that the down-regulation of c-Ski expression occurs at the protein level, not the mRNA level. Therefore, we investigated the posttranslational regulation of c-Ski in A10 cells and the rat balloon injury model. We demonstrated that an miRNA, miR-21, can directly bind to the 3′-UTR of c-Ski mRNA and negatively regulate c-Ski protein expression, showing that miR-21 is involved in the down-regulation of endogenous c-Ski after vascular injury. The small regulatory RNA miR-21 is encoded by a single gene and displays strong evolutionary conservation across a wide range of vertebrate species [24,25]. miR-21 plays a crucial role in a plethora of biological functions and diseases, including development, cancer, and inflammation [26–28]. More recently, the roles of miR-21 in
cardiovascular biology and disease have received significant attention [29,30]. Aberrant expression of miR-21 has been observed in these cardiovascular diseases [31,32]. For example, miR-21 was found to be significantly up-regulated in hypertrophic animal hearts [33] and in the cardiac fibroblasts of failing hearts [34]. In the early phase of acute myocardial infarction (AMI), miR-21 expression was significantly downregulated in infarcted areas but was up-regulated in border areas in infarcted rat hearts at 6 h and 24 h after AMI [35]. Ji et al. [36] reported that miR-21 expression increased more than five-fold in ballooninjured arteries compared with levels found in normal control vessels. In this study, we demonstrated that mature miR-21 expression in the arterial wall increased in a time-dependent manner after vascular injury in a rat balloon injury model. This finding displays the dynamic expression changes of miR-21 and indicates that miR-21 is involved in the progression of vascular injury. As more important functions of miR-21 have been revealed, potential target genes involved in miR-21-mediated cardiovascular effects have started to be identified. For instance, Thum et al. [34] reported the in vivo effect of miR-21 on cardiac hypertrophy, and Sayed et al. [37] identified that miR-21-mediated cardiomyocyte outgrowth was related to the target gene SPRY2. In ischemic heart disease, miR-21 was demonstrated to decrease myocardial infarct size. The protective effect of miR-21 against ischemia-induced cardiac cell death and myocardial infarction was mediated, at least in part, by the target gene programmed cell death 4 (PDCD4) and downstream molecules activator protein 1 (AP-1) [36] and heat-shock protein 70 (HSP70) [38,39]. During vascular injury progression, VSMC proliferation is considered to be a hallmark of intimal hyperplasia. In the present study, we found that c-Ski is a novel target gene of miR-21 in rat VSMCs. miR-21 may directly bind the 3′UTR of rat c-Ski mRNA to negatively regulate c-Ski protein expression and significantly suppress the inhibitory effect of c-Ski on rat VSMC proliferation. Supporting our findings, Ji et al. also reported that miR-21 has a stimulatory effect on VSMC proliferation and vascular neointimal growth. They found that phosphatase and tensin homology deleted from chromosome 10 (PTEN) [36] and PDCD4 [40] were two major target genes responsible for miR-21-mediated cellular effects on VSMCs. In combination, all of these data confirm the well-documented ability of one miRNA to exert its effect via multiple target genes.
Fig. 7. Effect of miR-21 on c-Ski-associated signal molecule expression changes in A10 cells. A10 cells were infected with the miR-21 mimic, inhibitor, NCs and/or Adc-Ski for 48 h. Protein was then extracted for western blot analysis for the downstream molecules of c-Ski in A10 cells. These molecules were previously demonstrated to be associated with the effect of c-Ski on A10 cell proliferation. (A) Western blot for p-p38. (B) Western blot for p21. (C) Western blot for p27.
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In healthy and diseased cells, it is evident that c-Ski expression levels strongly impact the molecular and cellular machinery of distinct cell types. c-Ski acts in combination with a number of cellular partners and, thus, has been shown to be involved in many signaling pathways, including those mediated by transforming growth factor β (TGFβ), nuclear hormone receptors, and Sonic hedgehog (Shh) [41–42]. Previously, we have demonstrated that c-Ski inhibition of VSMC proliferation largely depends on stimulating p38 activation to up-regulate the expression of downstream cyclin-dependent kinase inhibitors p21 and p27 [7]. Therefore, in the present study, we investigated whether p38–p21/p27 signaling was involved in the miR-21-mediated regulation of c-Ski and its effect on VSMC. The data confirmed that miR-21 negatively regulates c-Ski expression, leading to inhibition of p38 activation and p21/p27 expression, which, to some extent, results in the stimulation of VSMC proliferation. 5. Conclusion Taken together, our data significantly support three novel findings. First, we demonstrate that c-Ski is a novel target gene of miR-21 in rat VSMC. Second, we confirm that the miR-21-mediated negative regulation of c-Ski contributes to VSMC proliferation after vascular injury. Third, we attribute this role of miR-21 to inhibition of c-Ski–p38–p21/ p27 signaling. Conflict of interest All authors have no financial conflict of interest. Author's contributions Jun Li designed all experiments, performed the majority of all experiments and wrote the manuscript, Li Zhao performed the luciferase assay, Xie He and Ting Yang help in performing the proliferation assay. Kang Yang performed the data analysis. All authors read and approved the final version of the manuscripts. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81070229). References [1] R. Ross, N. Engl. J. Med. 340 (1999) 115–126. [2] A. Sachinidis, R. Locher, T. Mengden, W. Vetter, Biochem. Biophys. Res. Commun. 167 (1990) 353–359. [3] M.G. Davies, P.O. Hagen, Br. J. Surg. 81 (1994) 1254–1269. [4] S. Mani, H. Li, A. Untereiner, L. Wu, G. Yang, R.C. Austin, J.G. Dickhout, Š. Lhoták, Q.H. Meng, R. Wang, Circulation 127 (2013) 2523–2534.
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Contents lists available at ScienceDirect
Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
MiR-21 inhibits c-Ski signaling to promote the proliferation of rat vascular smooth muscle cells Jun Li a,⁎, Li Zhao b, Xie He b, Ting Yang b, Kang Yang a a b
Department of Cardiothoracic Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing 400038, China
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Article history: Received 5 December 2013 Accepted 18 December 2013 Available online 31 December 2013 Keywords: miR-21 c-Ski VSMC Proliferation
a b s t r a c t Previously, we reported that the decrease of endogenous c-Ski expression is implicated in the progression of vascular smooth muscle cell (VSMC) proliferation after arterial injury. However, the molecular mechanism of the down-regulation of c-Ski is not clear. In this study, a potential miR-21 recognition element was identified in the 3′-untranslated region (UTR) of rat c-Ski mRNA. A reporter assay revealed that miR-21 could recognize the miR-21 recognition element of c-Ski mRNA. In A10 rat aortic smooth muscle cells, overexpression of miR-21 significantly inhibited the expression of c-Ski protein and promoted cell proliferation, which could be blocked by inhibition of miR-21 or overexpression of c-Ski. Further investigation demonstrated that the effect of miR-21 on VSMC proliferation resulted from negative regulation of c-Ski to suppress p38–p21/p27 signaling, the downstream pathway of c-Ski in VSMCs. These results indicate that c-Ski is a target gene of miR-21. miR-21 specifically binds to the 3′-untranslated region of c-Ski and negatively regulates c-Ski expression to diminish the protective effects of c-Ski and stimulate VSMC proliferation in the progression of arterial injury. © 2014 Elsevier Inc. All rights reserved.
1. Introduction The proliferation of vascular smooth muscle cells (VSMCs) following vascular injury is critical to the pathogenesis of intimal hyperplasia and atherosclerosis [1–3]. More studies are focusing on looking into the endogenous factors that either stimulate VSMCs to proliferate or try to protect against proliferation. For example, some findings demonstrated that decreased endogenous production of hydrogen sulfide accelerates atherosclerosis, endogenous estrogen deficiency reduces proliferation and enhances apoptosis-related death in vascular smooth muscle cells and endogenous parathyroid hormone-related protein regulates the expression of PTH type 1 receptor and proliferation of vascular smooth muscle cells [4–6]. Recently, we have reported that c-Ski, a molecule expressed in VSMCs, suppresses VSMC stimulation and intimal hyperplasia in a rat balloon injury model [7]. However, the expression of endogenous cSki in the arterial wall and VSMCs was found to decrease in a timedependent manner after vascular injury. These findings indicate that down-regulation of endogenous c-Ski expression is implicated in the Abbreviations: VSMC, vascular smooth muscle cell; UTR, untranslated region; miRNAs, microRNAs; FBS, fetal bovine serum; AMI, acute myocardial infarction; PDCD4, programmed cell death 4; PDCD4, activator protein 1; HSP70, heat-shock protein 70; TGFβ, transforming growth factor β; Shh, Sonic hedgehog; PTEN, phosphatase and tensin homology deleted from chromosome 10. ⁎ Corresponding author at: Department of Cardiothoracic Surgery, Southwest Hospital, Third Military Medical University, 30 Gaotanyan, Shapingba, Chongqing 400038, China. Tel./fax: +86 23 68765330. E-mail address: [email protected] (J. Li). 0898-6568/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.12.013
progression of VSMC proliferation after arterial injury. These findings prompted us to further investigate the mechanism of c-Ski regulation. MicroRNAs (miRNAs) are a subset of small non-coding RNAs, averaging 22 nucleotides in length [8]. In mammals, the main function of mature miRNAs is thought to be recognizing the 3-untranslated regions of specific target mRNAs to suppress translation and, occasionally, to induce mRNA degradation [9,10]. Therefore, miRNAs are considered to be important negative regulatory factors. Growing evidence indicates that miRNAs control the processes of development and differentiation in many organisms, and miRNA dysregulation has been correlated with the aggressiveness and progression of several diseases [11–13]. Additionally, multiple miRNAs are found to be involved in VSMC proliferation and atherosclerosis by regulating gene expression [14–16]. Accordingly, in this study, we investigated the effect and mechanism of miRNAs in the regulation of c-Ski expression in VSMCs. We identified a potential miR-21 recognition element in the 3′-untranslated region (UTR) of rat c-Ski mRNA and confirmed that miR-21 could recognize the element and negatively regulate c-Ski protein expression. In addition, miR-21 stimulated VSMC proliferation via c-Ski inhibition. This effect mainly resulted from suppression of the c-Ski–p38–p21/p27 signaling pathway.
2. Materials and methods All animal procedures were carried out in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals Guidelines by the Third Military Medical University on the
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ethical use of animals and were approved by the State Science and Technology Commission of China. 2.1. Balloon injury model As previously described [7,17,18], male Sprague–Dawley rats (450–500 g) were anesthetized, and the left common carotid artery was exposed for a small arteriotomy. A 2 F Fogarty balloon catheter (Edwards Lifescience, U.S.A.) was introduced, and the balloon, inflated by a 1.6-atm pressure, was inserted and withdrawn three times. 2.2. Microarray and bioinformatics analysis At 28 days after balloon injury, the normal and injured vascular tissues were collected. MiRNA expression profiling was determined by miRNA microarray analysis using rat miRNA array probes. The bioinformatics analysis for potential miRNAs that may regulate c-Ski was performed online using database searches (miRBase, TargetScan, miRanda). 2.3. Cell culture Rat aortic A10 VSMCs were obtained from the American Tissue Culture Collection and grown, as recommended, in modified DMEM containing 4 mmol/L L-glutamine, 4.5 g/L glucose, 1 mmol/L sodium pyruvate, and 1.5 g/L sodium bicarbonate, supplemented with 10% fetal bovine serum (FBS) and antibiotics. 2.4. Western blot On days 0, 7, 14 and 28 post-injury, c-Ski expression in the rat carotid artery was detected by western blot following standard procedure. To detect the effect of miR-21 on c-Ski expression and downstream signaling, A10 cells were treated with 50 nM miR-21 mimic or inhibitor or were previously transfected with Adc-Ski to overexpress c-Ski, as previously described [7]. Twelve hours after treatment, western blot analysis was performed to determine protein expression using antibodies against c-Ski (sc-33693, Santa Cruz), p-p38 (sc-365846, Santa Cruz), p21 (sc397, Santa Cruz) and p27 (sc 528; Santa Cruz). In these experiments, β-actin (sc-1616; Santa Cruz) served as the endogenous control. 2.5. Real-time RT-PCR for mature microRNA For the quantification of mature miR-21, polyadenylation and reverse transcription were performed using the All-in-One TM miRNA qRT-PCR Detection Kit (GeneCopoeia) according to the manufacturer's protocol. Quantitative PCR was then carried out with SYBR green detection, with U6 used as an internal control for template normalization. The primers for mature miR-21 and U6 were both provided by GeneCopoeia, Inc. 2.6. Luciferase assays The rat c-Ski mRNA fragment from 2218 to 2786 (No. XM233731) containing the miR-21 recognition element was commercially synthesized at Sunbio Medical Biotech (Shanghai, China) and cloned into the pMIR-REPORT vector (named pMIR-c-Ski) after digestion with XhoI and HindIII. The primers were 5′-CTGGAGAAGGTGGTGAAGGA-3′ (forward) and 5′-CACGATGCGAGGAGTG ATGT-3′ (reverse). The pMIR-cSki-mutant plasmid, where the miR-21 recognition element was mutated to “GCTGATCG,” was generated using the QuikChange site-directed mutagenesis kit (Stratagene, CA). Subsequently, A10 cells were transiently transfected using Lipofectamine2000 with luciferase reporter pMIR-REPORT, pMIR-c-Ski or pMIR-c-Ski mutant. The reporter constructs were transiently transfected with 50 nM miR-21 mimic, 50 nM miR-21 inhibitor or negative control. Transfection efficiency was
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monitored by co-transfection of pMIR-REPORT-β-gal. Cell extracts were prepared after transfection, and luciferase and β-galactosidase assays were performed according to the manufacturer's instruction. All transfection data were expressed as the ratios of luciferase activities/ β-galactosidase activities. 2.7. Cell proliferation assay Proliferation rates of A10 cells were determined by the Cell Count Kit-8 (CCK-8 Kit, Beyotime Inst Biotech, China) using the WST-8 (2(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2H-tetrazolium (monosodium salt)) dye method according to the manufacturer's instructions and references [7]. 2.8. Statistical analysis The values are expressed as the means ± SEM. An unpaired Student's t-test was used to evaluate the significant differences between the control and treated groups. Values of P b 0.05 were considered to be significant. All experiments were repeated at least three times. 3. Results 3.1. Endogenous c-Ski protein expression, rather than mRNA level, decreases in a time-dependent manner after vascular injury In the rat balloon injury model, endogenous c-Ski protein and mRNA levels were detected. As shown in Fig. 1A, no significant changes in c-Ski mRNA expression levels were observed during the 28 days postvascular injury. However, western blot results showed that 7 days after vascular injury, c-Ski protein expression decreased compared with the uninjured group. On days 14 and 28 post-injury, the decreases in c-Ski protein expression were significant (Fig. 1B). These data demonstrate that endogenous c-Ski protein, rather than mRNA, expression in the injured rat carotid arteries was significantly down-regulated in a time-dependent manner. These results indicate that c-Ski expression may be post-transcriptionally regulated. 3.2. miR-21 shows potential to regulate c-Ski via microarray and database searches To investigate whether c-Ski is modulated by miRNA, an important mechanism of post-transcriptional regulation, we assayed significantly changed miRNAs after vascular injury by microarray and investigated what potential miRNAs regulate c-Ski via online database searches (miRBase, TargetScan, miRanda). Compared with levels in normal control vessels, 8 miRNAs were found to have more than a five-fold increase in expression levels in balloon-injured arteries. However, among the aberrantly expressed miRNAs, only miR-21 was indicated to have the potential to regulate c-Ski by bioinformatics analysis (Fig. 2A). The potential miR-21 recognition element is located on +2516 to +2523 in the 3′-UTR of rat c-Ski mRNA (Fig. 2B). 3.3. Endogenous miR-21 levels in the arterial wall increase in a timedependent manner after vascular injury Using the rat balloon injury model, we then assayed the endogenous miR-21 expression levels on days 0, 7, 14 and 28 after vascular injury. Contrary to c-Ski protein expression levels, endogenous miR-21 levels in the arterial wall increased in a time-dependent manner during the 28 days following vascular injury (Fig. 3). These data further indicate that miR-21 may be involved in the down-regulation of c-Ski protein expression after vascular injury.
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Fig. 1. Time course of endogenous c-Ski expression after vascular injury. The c-Ski mRNA and protein expression levels in injured rat carotid arteries were detected on days 0, 7, 14 and 28 post-balloon angioplasty in rats. (A) RT-PCR for c-Ski mRNA expression in injured rat carotid arteries. (B) Western blot for c-Ski protein expression in injured rat carotid arteries. The representative results are from 3 separate experiments, and the data from RT-PCR experiments are expressed as the mean ± S.E.M.
3.4. miR-21 regulates c-Ski protein expression in A10 cells The effects of miR-21 on c-Ski protein levels in A10 cells were examined by western blot. A10 cells were transfected with an miR-21 mimic, a synthetic RNA duplex that mimics endogenous miR-21, which significantly increased miR-21 levels (Fig. 4A). This transfection induced a repression of c-Ski protein levels compared with the control (Fig. 4C). Conversely, transfection of the miR-21 inhibitor, a single-stranded modified RNA with a complimentary sequence to the mature miRNA that functions by sequestering/degrading endogenous miRNAs, markedly decreased miR-21 expression in A10 cells while subsequently significantly inducing c-Ski protein levels (Fig. 4B and C). 3.5. Validation of the miR-21 target site in the 3′-UTR of c-Ski mRNA Next, using a luciferase reporter assay, we tested whether miR-21 regulated c-Ski in A10 cells through direct targeting of the c-Ski 3′UTR. As shown in Fig. 5, the luciferase activity of pMIR-c-Ski plasmid, in which the miR-21 recognition element was inserted downstream of the luciferase gene, was down-regulated to some extent in the presence of the NC of the miR-21 mimic or inhibitor compared with the control (pMIR-REPORT). Because miR-21 is highly expressed in VSMCs, this may result from the effect of endogenous miR-21 on the luciferase activity of pMIR-c-Ski plasmid. It should be noted that, compared with the treatments of the NCs, the miR-21 mimic significantly decreased the luciferase activity of pMIR-c-Ski (Fig. 5A), while the miR-21 inhibitor restored the repression of luciferase activity of pMIR-c-Ski induced by endogenous miR-21 (Fig. 5B). However, these effects of the miR-21 mimic and inhibitor on the luciferase activity of the pMIR-c-Ski mutant
Fig. 2. Potential miRNA to regulate rat c-Ski expression. On day 28 after balloon injury, miRNAs with greater than a five-fold increase in expression were detected by miRNA microarray. Potential miRNAs to regulate c-Ski were identified by online database searches. (A) The intersection of miRNA microarray data and bioinformatics analysis online. (B) The potential binding site for miR-21 in the 3′-UTR of rat c-Ski mRNA.
plasmid (in which the miR-21 recognition element was mutated) were not observed (Fig. 5A and B). These data determine that miR-21 binds to the putative miR-21 target site within the c-Ski 3′-UTR in A10 cells. 3.6. miR-21 promotes the proliferation of A10 cells via regulation of c-Ski Our previous study reported that c-Ski plays a role in inhibiting VSMC proliferation [7]. Accordingly, to explore whether the miR-21mediated regulation of c-Ski affects the growth of VSMCs, the proliferation of A10 cells was investigated. As shown in Fig. 6, compared with the control, the miR-21 mimic stimulated, while the miR-21 inhibitor suppressed, A10 cell proliferation significantly. However, in A10 cells overexpressing c-Ski, the effect of the miR-21 mimic on the induction of cell proliferation was largely blocked, while the inhibitory role of the miR21 inhibitor was enhanced. These results suggest that miR-21 promotes the proliferation of A10 cells via regulation of c-Ski. 3.7. miR-21 modulation of c-Ski affects the expression of p38, p21 and p27 Because p38 and p21/p27 expression was demonstrated to be downstream of c-Ski and involved in mediating the inhibitory effect of c-Ski on VSMC proliferation, we then assayed the effect of miR-21 on p38–p21/p27 signaling. Western blot results showed that the miR-21 mimic suppressed, while the miR-21 inhibitor enhanced, p38 phosphorylation (Fig. 7A) and p21/p27 expression levels (Fig. 7B and C). The effects of the miR-21 mimic could be blocked both by the miR-21 inhibitor and the overexpression of c-Ski (Fig. 7). However, the NCs of the miR-21 mimic and miR-21 inhibitor had no effect on these molecules. These data confirm that miR-21 targets c-Ski to modulate the p38– p21/p27 signaling involved in the effect of miR-21 on VSMC proliferation, as described above.
Fig. 3. Time-course of endogenous miR-21 expression after vascular injury. The expression levels of miR-21 in injured rat carotid arteries were detected by RT-PCR on days 0, 7, 14 and 28 post-balloon angioplasty. The representative results are from 3 separate experiments, and the data are expressed as the mean ± S.E.M. * Indicates P b 0.01 compared to day 0.
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Fig. 4. Effects of overexpression or inhibition of miR-21 on c-Ski protein levels in A10 cells. The miR-21 mimic (50 nM), inhibitor (50 nM) or negative control was transfected into A10 cells. After 48 h, the cells were harvested and total RNA and protein extracts were isolated. (A) and (B) Levels of mature miR-21 expression were determined by RT-PCR. The values shown represent the mature miR-21 levels normalized to U6 snRNA levels, relative to controls. (C) c-Ski protein expression was determined by western blot. The representative results are from 3 separate experiments, and the data are expressed as the mean ± S.E.M. * Indicates P b 0.01 compared to the control.
4. Discussion c-Ski was originally discovered as the cellular homolog of retroviral v-Ski and was thus classified as a proto-oncogene [19,20]. In addition
to this, numerous recent studies have found more and more important functions of c-Ski in physiological and pathological conditions, such as in normal development and wound healing [21–23]. We previously reported that c-Ski has an inhibitory effect on VSMC proliferation and
Fig. 5. Validation of miR-421 target sites via luciferase reporter system. Reporter constructs were transiently transfected into A10 cells with 50 nM miR-21 mimic, inhibitor or their negative controls (NC), respectively. The relative luciferase activities were calculated as luciferase activities/β-galactosidase activities. (A) Luciferase activities in the presence of the miR-21 mimic. (B) Luciferase activities in the presence of the miR-21 inhibitor. The representative results are from 3 separate experiments, and the data are expressed as the mean ± S.E.M. * Indicates P b 0.01 when comparing the two groups.
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Fig. 6. Effect of miR-21 on A10 cell proliferation. The proliferation of A10 cells transfected with the miR-21 mimic or inhibitor was assayed using a CCK-8 Kit with the WST-8 dye method at 0 h, 24 h, 48 h and 72 h after transfection. In addition, to detect whether regulation of c-Ski was involved in the effect of miR-21, c-Ski was overexpressed in rat A10 cells by Adc-Ski transfection, as previously described. The representative results are from 3 separate experiments, and the data are expressed as the mean ± S.E.M. * Indicates P b 0.01 compared with the control; # Indicates P b 0.01 when comparing the two groups.
intimal hyperplasia in a rat balloon injury model. We also found that endogenous c-Ski expression in the arterial wall decreased in a timedependent manner after vascular injury [7]. In this study, we further showed that the down-regulation of c-Ski expression occurs at the protein level, not the mRNA level. Therefore, we investigated the posttranslational regulation of c-Ski in A10 cells and the rat balloon injury model. We demonstrated that an miRNA, miR-21, can directly bind to the 3′-UTR of c-Ski mRNA and negatively regulate c-Ski protein expression, showing that miR-21 is involved in the down-regulation of endogenous c-Ski after vascular injury. The small regulatory RNA miR-21 is encoded by a single gene and displays strong evolutionary conservation across a wide range of vertebrate species [24,25]. miR-21 plays a crucial role in a plethora of biological functions and diseases, including development, cancer, and inflammation [26–28]. More recently, the roles of miR-21 in
cardiovascular biology and disease have received significant attention [29,30]. Aberrant expression of miR-21 has been observed in these cardiovascular diseases [31,32]. For example, miR-21 was found to be significantly up-regulated in hypertrophic animal hearts [33] and in the cardiac fibroblasts of failing hearts [34]. In the early phase of acute myocardial infarction (AMI), miR-21 expression was significantly downregulated in infarcted areas but was up-regulated in border areas in infarcted rat hearts at 6 h and 24 h after AMI [35]. Ji et al. [36] reported that miR-21 expression increased more than five-fold in ballooninjured arteries compared with levels found in normal control vessels. In this study, we demonstrated that mature miR-21 expression in the arterial wall increased in a time-dependent manner after vascular injury in a rat balloon injury model. This finding displays the dynamic expression changes of miR-21 and indicates that miR-21 is involved in the progression of vascular injury. As more important functions of miR-21 have been revealed, potential target genes involved in miR-21-mediated cardiovascular effects have started to be identified. For instance, Thum et al. [34] reported the in vivo effect of miR-21 on cardiac hypertrophy, and Sayed et al. [37] identified that miR-21-mediated cardiomyocyte outgrowth was related to the target gene SPRY2. In ischemic heart disease, miR-21 was demonstrated to decrease myocardial infarct size. The protective effect of miR-21 against ischemia-induced cardiac cell death and myocardial infarction was mediated, at least in part, by the target gene programmed cell death 4 (PDCD4) and downstream molecules activator protein 1 (AP-1) [36] and heat-shock protein 70 (HSP70) [38,39]. During vascular injury progression, VSMC proliferation is considered to be a hallmark of intimal hyperplasia. In the present study, we found that c-Ski is a novel target gene of miR-21 in rat VSMCs. miR-21 may directly bind the 3′UTR of rat c-Ski mRNA to negatively regulate c-Ski protein expression and significantly suppress the inhibitory effect of c-Ski on rat VSMC proliferation. Supporting our findings, Ji et al. also reported that miR-21 has a stimulatory effect on VSMC proliferation and vascular neointimal growth. They found that phosphatase and tensin homology deleted from chromosome 10 (PTEN) [36] and PDCD4 [40] were two major target genes responsible for miR-21-mediated cellular effects on VSMCs. In combination, all of these data confirm the well-documented ability of one miRNA to exert its effect via multiple target genes.
Fig. 7. Effect of miR-21 on c-Ski-associated signal molecule expression changes in A10 cells. A10 cells were infected with the miR-21 mimic, inhibitor, NCs and/or Adc-Ski for 48 h. Protein was then extracted for western blot analysis for the downstream molecules of c-Ski in A10 cells. These molecules were previously demonstrated to be associated with the effect of c-Ski on A10 cell proliferation. (A) Western blot for p-p38. (B) Western blot for p21. (C) Western blot for p27.
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In healthy and diseased cells, it is evident that c-Ski expression levels strongly impact the molecular and cellular machinery of distinct cell types. c-Ski acts in combination with a number of cellular partners and, thus, has been shown to be involved in many signaling pathways, including those mediated by transforming growth factor β (TGFβ), nuclear hormone receptors, and Sonic hedgehog (Shh) [41–42]. Previously, we have demonstrated that c-Ski inhibition of VSMC proliferation largely depends on stimulating p38 activation to up-regulate the expression of downstream cyclin-dependent kinase inhibitors p21 and p27 [7]. Therefore, in the present study, we investigated whether p38–p21/p27 signaling was involved in the miR-21-mediated regulation of c-Ski and its effect on VSMC. The data confirmed that miR-21 negatively regulates c-Ski expression, leading to inhibition of p38 activation and p21/p27 expression, which, to some extent, results in the stimulation of VSMC proliferation. 5. Conclusion Taken together, our data significantly support three novel findings. First, we demonstrate that c-Ski is a novel target gene of miR-21 in rat VSMC. Second, we confirm that the miR-21-mediated negative regulation of c-Ski contributes to VSMC proliferation after vascular injury. Third, we attribute this role of miR-21 to inhibition of c-Ski–p38–p21/ p27 signaling. Conflict of interest All authors have no financial conflict of interest. Author's contributions Jun Li designed all experiments, performed the majority of all experiments and wrote the manuscript, Li Zhao performed the luciferase assay, Xie He and Ting Yang help in performing the proliferation assay. Kang Yang performed the data analysis. All authors read and approved the final version of the manuscripts. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81070229). References [1] R. Ross, N. Engl. J. Med. 340 (1999) 115–126. [2] A. Sachinidis, R. Locher, T. Mengden, W. Vetter, Biochem. Biophys. Res. Commun. 167 (1990) 353–359. [3] M.G. Davies, P.O. Hagen, Br. J. Surg. 81 (1994) 1254–1269. [4] S. Mani, H. Li, A. Untereiner, L. Wu, G. Yang, R.C. Austin, J.G. Dickhout, Š. Lhoták, Q.H. Meng, R. Wang, Circulation 127 (2013) 2523–2534.
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