miR-132-3p mediates Functions of Vascular Smooth Muscle Cells through targeting PTEN expression and ERK1/2 pathway


 Background

This study was aimed to investigate the functional role of the microRNA (miR)-132-3p in rat vascular smooth muscle cells (VSMCs) and the potential mechanisms in abdominal aortic aneurysm (AAA).
Methods

VSMCs were transfected with miR-132-3p mimics and inhibitors, and the effects of miR-132-3p on VSMCs proliferation, migration were assessed by CCK-8 assay and Boyden chamber cell invasion assay, respectively. miRNA targets were determined using bioinformatics and luciferase reporter assays. The protein expression of phenotype markers and related signaling pathways were detected by Western blot.
Results

Overexpression of miR-132-3p in VSMCs attenuated VSMCs proliferation and migration. Conversely, the opposite effect was obtained with the inhibition of miR-132-3p. We further demonstrated that miR-132 could significantly promote the expression of VSMCs marker genes ACTA2 and MYH1. Reporter assays and western blot validated that PTEN as a direct target of miR-132-3p in VSMCs. Besides, miR-132-3p overexpression could also promote the expression of ras and c-myc, and activate the phosphorylation levels of ERK1/2.
Conclusions

These results indicate that miR-132-3p is a critical regulator in maintaining normal functions of VSMCs through PTEN-ERK1/2 axis. Restoring expression of miR-132-3p may serve as a potential therapeutic approach for treatment of AAAs.

Abdominal aortic aneurysm (AAA) rupture is one of the major causes of death in elderly people [1,2].
Although current frontline therapy options such as surgery and endovascular treatment have been improved greatly in recent years, the molecular mechanism responsible for AAA formation is still unclear, which makes the development of medical therapies much slower 3. Aneurysm formation is a complicated multifactorial progressive process involving destructive remodeling of the connective tissue around the affected segment of the aorta wall, characterized by dysfunctions in the vascular smooth muscle cells(VSMCs) which may be responsible for the progression of AAAs [2,3,4].
MicroRNAs (miRNAs) are single-stranded RNA molecules of approximately 19-25 nucleotides in length that can mediate multiple cellular processes, including proliferation, differentiation and apoptosis [5,6]. A number of normally expressed miRNAs have been reported in the cardiovascular system, to attune VSMCs phenotypes and functions and to be abnormally expressed in vascular diseases [7][8][9][10][11]. miRNAs could make a precise regulation of the intricate molecular networks by modulating mRNA and/or protein levels at the post-transcriptional level.
miR-132-3p was rst identi ed with a 7-fold high expression in a rat AAA model in our previous study, but its role in AAAs formation is not fully understood [10]. Therefore, further study of the effect of miR-132-3p on VSMCs functions will help us understand its molecular mechanism in the formation of AAAs and develop new diagnostic and therapeutic approaches. In the present study, we demonstrated that miR-132-3p could mediate VSMCs function, activate ERK1/2 signaling pathway and directly target phosphatase and tensin homolog (PTEN), which might be a new therapeutic target for AAAs.
The miR-132-3p mimic, inhibitor and control were transfected into VSMCs, and the expression of miR-132-3p was veri ed by qRT-PCR. Figure 1 (A) shows that the level of miR-132-3p expression was signi cantly downregulated in cells transfected with the miR-132-3p inhibitor. When the miR-132-3p mimic was transfected, the miR-132-3p level was remarkably increased compared to that in the control cells (Fig. 1B). A CCK-8 assay demonstrated that downregulation of miR-132-3p promoted cell proliferation, while the miR-221-3p mimic impaired cell proliferation on the contrary. (Fig. 1C) Moreover, inhabitation of miR-132-3p promoted VSMCs migration as assessed by Boyden chamber assays ( Fig. 1D). In contrast, overexpression of miR-132-3p in VSMCs signi cantly attenuated VSMCs migration ( Fig. 1E). Together, these data demonstrate that miR-132-3p is a potent inhibitor for VSMCs proliferation and migration.
miR-132-3p regulates the expression of phenotype marker genes The modulation between a contractile phenotype and synthetic phenotype in smooth muscle cells was associated with changes in proliferation ability, migration capability, and expression level of VSMCs marker genes 11 . Smooth muscle a-actin (ACTA2) and smooth muscle myosin heavy chain (MYH11) were changed markedly during VSMCs phenotype modulation 12,13 . To reveal the role of miR-132-3p in regulating the expression of VSMCs marker genes, we transiently transfected rat VSMCs with miR-132-3p mimics or inhibitors for 72 h. The expression levels of marker genes in VSMCs were examined by western blotting with ACTIN as the internal reference. The results showed that miR-132-3p downregulated the expression of contractile phenotype marker genes ( Fig. 2A), while it upregulated that of synthetic phenotype marker genes (Fig. 2B). Our ndings showed that overexpression of miR-132-3p by mimics signi cantly attenuated contractile marker expression in cultured VSMCs.
PTEN was a target of miR-132-3p Among predicated candidate genes, we found that the gene encoding PTEN harbored a potential miR-132-3p binding site. The alignment of miR-132-3p and the 3'-UTR of PTEN was shown of Fig. 3A. Next, the wild type or mutant 3'UTR of PTEN gene was cloned and inserted into pMIR reporter vector. The results showed that overexpression of miR-132-3p led to a diminish of the luciferase activity carrying the wild-type PTEN 3'UTR but had noticeable effect on the luciferase reporter with mutated PTEN 3′-UTR (Fig. 3B). Transfection of miR-132 mimic also decreased the protein amount of PTEN (Fig. 3C, D). Conversely, down-regulation of miR-132-3p inhibitor signifcantly increased the expression levels of PTEN ( Fig. 3C, D). Therefore, our results indicated that PTEN might be a target of miR-132-3p in VSMCs.
Overexpression of miR-132-3p activates ERK 1/2 signaling pathway In this study, we also explored whether ERK signaling pathway activation was affected by miR-132-3p.

Discussion
Alterations of VSMCs structural and functional connectivity have previously been described to play a crucial role in the initiation and progression of cardiovascular diseases, such as atherosclerosis obliteran, restenosis, and hypertension [11,14]. VSMCs phenotypes can be switched to each other reversibly, and once irreversible transformation occurs, vascular disease occurs [15]. It has been shown that phenotypes of VSMCs change from the contractile to the synthetic phenotype contribute to atherosclerosis, aortic aneurysms, and neointimal proliferation [16,17]. Therefore, therapeutic strategies that regulate abnormal VSMCs phenotypic alterations hold great promise for impeding the progression of vascular diseases.
Previous studies demonstrated that miRNAs participated in regulating VSMCs phenotype alterations [18].
In line with these studies, our in vitro experiments further indicated that upregulation of miR-132-3p in VSMCs strongly upgraded the expression levels of SMC-speci c genes, while down-regulation of miR-132-3p had the reverse effect on these markers. In order to investigate the molecular mechanism of microRNA-132-3p in VSMCs function regulation, we identi ed PTEN as a direct downstream target of miR-132-3p PTEN is a member of type-I protein tyrosine phosphatase (PTP) family, originally found as a tumorsuppressed gene, regulating tumorigenic functions, such as apoptosis, cell cycle, cell adhesion, and cell migration [19,20]. Current evidence showed that it could inhibit the cell proliferation and migration in VSMCs [21,22]. In our study, we con rmed that overexpression of miR-132-3p signi cantly represses PTEN expression in rat VSMCs and validated the transcription factor PTEN as a direct target of miR-132-3p in VSMCs. Additionally, ectopic expression of miR-132-3p inhibited cell proliferation and decelerated VSMCs migration. Therefore, we may conclude miR-132-3p mediate the function and phenotype change of VSMCs via PTEN.
Extracellular signal-regulated kinase (ERK) 1/2signaling pathway plays a crucial role in phenotypic modulation in VSMCs [23], regulating both contractile [24] and synthetic markers [25].It has also been found highly expressed in AAA tissues, which is an important modulator of MMPs during AAA formation [26,27].Interestingly,ERK1/2 pathway was negatively regulated as an important downstream target of PTEN in many diseases [28,29]. Similarly, our results revealed that miR-132-3p could impede PTEN expression and promote ERK phosphorylation along with its downstream target genes activation (c-myc, ras). Therefore, these ndings suggest that the inhibitory effects of overexpressed miR-132-3p on AAA formation likely result from a combination of its ability to target both PTEN and ERK1/2 pathway.
It should be noted that there are some limitations to our study. There were more than a hundred predicted targets for miR-132-3p according to the website tools used. Whether these unexamined target genes could participate in miR-132-3p mediated VSMCs function is unknown. Even among the con rmed genes in this study, their interactions are still unclear. These limitations will hopefully be addressed in future studies.

Conclusions
In conclusion, miR-132-3p serves as an important regulator of VSMCs proliferation, migration and promotes a VSMCs synthetic phenotype modulation through targeting PTEN-ERK1/2 axis. Therefore, restoring miR-132-3p expression in the VSMCs may become an attractive therapeutic approach for treatment of AAA. Statistical analysis × Data were expressed as the means ± standard deviations. A student's t test or one-way ANOVA was used to compare variables. Differences were considered to be statistically signi cant at P-values < 0.05. All statistical analyses were performed using the SPSS statistical package (version 18.0; SPSS Inc., Chicago, Illinois).

Declarations
Ethics approval and consent to participate This experimental research on SD Rat cells was complied with the revised Animals (Scienti c Procedures) Act 1986 in the UK and Directive 2010/63/EU in Europe) and was approved with Shanghai Ninth people's Hospital ethics committee.

Consent for publication Not Applicable
Availability of data and materials Materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes, without breaching participant con dentiality.

Competing interests
The authors declare that they have no competing interests.