Effect of MicroRNA-30e on the Behavior of Vascular Smooth Muscle Cells via Targeting Ubiquitin-Conjugating Enzyme E2I

Abstract

Background: Many microRNAs (miRNAs) have recently been shown to demonstrate critical roles in differentiation, proliferation and migration of vascular smooth muscle cells (VSMCs).

Methods and Results: In this study, a certain amount of miRNA expression in VSMCs was evaluated by real-time polymerase chain reaction, and it was found that microRNA-30e (miR-30e) was expressed more strongly than other common vascular well-expressed miRNAs in vitro. Subsequently, both a gain and loss of function study was performed in vitro and in vivo. It was found that miR-30e in VSMCs was strongly downregulated concomitantly with stimulation, and miR-30e inhibited VSMCs proliferation and migration both in vitro and in vivo. Furthermore, ubiquitin-conjugating enzyme E2I (Ube2i) was identified as the target gene of endogenous miR-30e by luciferase reporter assay, and it was confirmed that overexpression of miR-30e significantly reduced Ube2i and inhibited the phenotypic switch of VSMCs. Knockdown of Ube2i had an influence over the proliferation and migration of cultured VSMCs, as same as the miR-30e mimic did. Overexpression of miR-30e induced the apoptosis of VSMCs and deregulated the protein expression of IkBα, which is crucial for the NFκB signal pathway.

Conclusions: The results of this study indicated that miR-30e in VSMCs exerted an anti-atherosclerosis effect via inhibiting proliferation and migration, and promoting apoptosis of VSMCs. More specifically, it was demonstrated that miR-30e exhibited these effects on VSMCs partially through targeting Ube2i and downregulating the IκBα/NFκB signaling pathway.

Cardiovascular diseases have long been recognized as the leading contributors to the death rate in both industrialized societies and developing countries.^1,2 Clinically, neointima is a common pathological lesion in a multitude of cardiovascular diseases such as atherosclerosis, coronary artery diseases, postangioplasty restenosis, and transplantation arteriopathy, as a result of the migration and proliferation of vascular smooth muscle cells (VSMCs) from the media. Aberrant VSMCs are pivotal for the pathogenesis of these diseases. Contrary to other terminally differentiated cells, VSMCs can proliferate and migrate in response to the altered homeostasis of vasculature,^3,4 which is normally maintained in quiescence and equilibrium, whereas the capacities of VSMCs to proliferate and migrate are markedly enhanced by various growth factors and cytokines such as platelet-derived growth factor-BB (PDGF-BB), fibroblast growth factor, insulin-like growth factor-1, and tumor necrosis factor-α (TNF-α),^5,6 as well as a number of genes and cytokines that have been identified as risk factors in these processes. Intriguingly, microRNAs (miRNAs) are functional transcription factors and have been implicated in the regulation of the proliferation and migration of VSMCs.^7,8

MiRNAs are small, non-coding RNA molecules and negative regulators of gene expression via the degradation or translational inhibition of their target mRNAs,^9,10 which involve a variety of pathological processes of atherosclerosis, leukemias, lung cancer, colorectal cancer, diabetes mellitus, etc.^11–13 The expression of miRNAs are tissue- and cell-specific, involving a panorama of cell processes such as proliferation, development, migration, differentiation and apoptosis. As a group, miRNAs directly regulate at least 30% of the genomes in cells. Many miRNAs have recently been found to be critical for VSMC differentiation, proliferation and migration.^7,8 It is likely that many others are involved in controlling such processes. Our goal was to identify the miRNAs involved in regulating the behavior of VSMCs.

MiR-1 is highly expressed both in cardiac and skeletal muscles, and is encoded by 2 genes (miR-1-1 and miR-1-2), each of which is co-expressed bicistronically with 1 copy of the 2 miR-133a genes.¹⁴ This miRNA plays a key role in differentiation and proliferation of muscle cells.¹⁵ MiR-1 has also been shown to participate in VSMC differentiation and phenotypic regulation.¹⁶

Many studies have indicated that the knockdown of microRNA-30e (miR-30e) substantially represses lung cancer cell growth and tumorigenicity in a dose-dependent manner,¹⁷ and antagonizes the morphological changes and the migratory and invasive characteristics of carcinoma cells.¹⁸ Other studies have shown miR-30e involvement in cancer cell proliferation, migration and invasion.^19–23 This research suggests that this miRNA could be a critical controller of cell proliferation, migration and differentiation more generally. However, its biological function in VSMCs remains unclear. We considered whether miR-30e is expressed abundantly in VSMCs and whether miR-30e could modulate the behavior of VSMC; consequently, what the regulation mechanism of miR-30e in VSMCs was.

We predicted targets of miR-30e, in particular, at cell proliferation and migration by computational analysis. The analysis suggested that miR-30e may be bound to the 3’-UTR of ubiquitin-conjugating enzyme E2I (Ube2i) mRNA. Ube2i is an E2-conjugating enzyme essential for sumoylation, which was first identified as a human homolog of yeast UBC9 in a yeast two-hybrid system, which interacted with RAD52 and RAD51.²⁴ Ube2i modified several proteins and signal pathways;²⁵ and also promoted cell proliferation and migration.^26–28 We proposed that Ube2i was involved in the regulation mechanism of miR-30e, and we aimed to investigate the role of Ube2i in the panorama of VSMC behavior.

In this study, we were determined to elucidate the effects and the target of miR-30e in the process of the proliferation, migration and apoptosis of VSMCs.

Methods

VSMC Culture and Carotid Artery Balloon Injury in Rats

The studies were approved by the Animal Ethics Committee of the Xuzhou Medical University (Number CMCACUC2012-04-078). All animal experiments and procedures were carried out in accordance with the approved guidelines. VSMCs were isolated from normal aorta of adult male rats (Xuzhou Medical University) by the tissue adherent method. The cells were used for the third and fourth passages. The carotid artery balloon injury model was described in our previous study.²⁹

Oligo Transfection in Cultured VSMCs and Adenovirus Transduction in Injured Carotid Artery

The miR-30e and miR-1 mimics or inhibitors (RiboBio) were added to the complexes for overexpressing or knocking down the miRNAs in vitro. The experiments in vitro were performed in the following groups: control group (CTR group), miR-30e mimic group, miR-30e inhibitor group, miR-30e mimic control group (miR-CTR group), miR-1 mimic group, miR-1 inhibitor group. Ube2i expression was knocked down using Ube2i siRNA (RiboBio) in vitro. Cells were transfected using a RiboFect^TM CP Transfection Kit (RiboBio) according to the manufacturer’s protocol.

Rats were randomly selected for the control group (CTR group), miR-30e group, miR-30e antago group and the miR-30e scramble group (miR-CTR group) (each group, n=9). Adenovirus of miR-30e, miR-30e antago and adenovirus of miR-30e scramble (Hanbio) were transduction in injured carotid artery; the protocol of this experiment is described in a previous study.³⁰ Rats were provided with water and food provided ad libitum for 14 days.

Results

Evaluation of the RNA Expressions of the miR-30 Family and Known Vascular Well-Expressed miRNAs

The levels of the miR-30 family were detected by RT-PCR separately. miR-30e is a member of the miR-30 family and they share similar targets: miR-30a/b/c/d/e and miR-384-5p. The levels of miR-30e were much greater than that of other miR-30 family members in VSMCs (Figure 1A). As there were no significant differences of the RNA expression between the control groups and the miR-control groups,³¹ we detected the levels of RNA expression of the miR-30 family and some other vascular well-expressed miRNAs in these four groups: the CTR group, the miR-CTR group, the miR-30e mimic group and the miR-30e inhibitor group; the latter 3 groups were transfected with miRNAs and incubated with platelet-derived growth factor (PDGF)-BB (20 ng/mL). After stimulation by PDGF-BB, the expression of miR-30e RNA in the miR-30e mimic group was 8-fold higher than that of other groups, while in the miR-CTR and the miR-30 inhibitor group, miR-30e decreased by one-half and decreased to one-fifth compared with the CTR group (Figure 1B). Both miR-30a–d and 384-5p did not change among the four groups. (Figure 1C–G). As shown in Figure 1H and I, we compared miR-30e with miR-1 and other known vascular well-expressed miRNAs, including miR-221, miR-222 and miR-200b, miR-200c.

Figure 1.

Evaluation of the miR-30 family and known vascular well-expressed microRNAs in vascular smooth muscle cells (VSMCs). (A) miR-30e was the most expressed miR-30 family member in VSMCs. ***P<0.001 vs. miR-30e; ^#P<0.05, ^###P<0.001 vs. miR-30a; ^&&P<0.01, ^&&&P<0.001 vs. miR-30b; ^$P<0.05, ^$$$P<0.001 vs. miR-30c; ^@@@P<0.001 vs. miR-30d. (B) miR-30e decreased after incubation with platelet-derived growth factor-BB (PDGF-BB); miR-30e mimic and inhibitor exerted their effects. ***P<0.001 vs. Control (CTR); ^###P<0.001 vs. miR-CTR; ^&&&P<0.001 vs. miR-30e mimic. ***P<0.001 vs. the CTR group; ^###P<0.001 vs. the miR-CTR; ^&&&P<0.001 vs. the miR-30e mimic. (C–G) miR-30a–d and 384-5p did not alter after transfection and incubation. (H,I) The expression of miR-30e in VSMCs was approximately close to that of miR-221 levels. Expression of miRNA-200b/c compared to miR-30e expression in rat VSMCs. *P<0.05, ***P<0.001 vs. miR-30e; ^#P<0.05 vs. miR-1. Data are expressed as the mean±SD (n=3).

MiR-30e Regulation in Quiescent and Proliferating VSMCs In Vitro

To investigate the potential link between VSMC proliferation and miRNA expression, miRNA expression levels in VSMCs were measured at different times of proliferation. The VSMCs were stimulated with 10% serum at 0, 12, 24, 48 and 96 h after synchronization by 24-h serum-starvation-induced quiescence. Their proliferation was detected by Edu dyeing. At 24 h, the proliferating VSMCs were most numerous (Figure 2A,B). At the different time points after serum stimulation, miR-30e was significantly downregulated in the proliferating VSMCs compared with their quiescent counterparts at 0 h (Figure 2C). In contrast, the miR-1 levels remained unchanged (Figure 2D). Proliferating VSMCs have very low levels of miR-30e.

Figure 2.

miR-30e regulation in quiescent and prolife-rating proliferating and migrating vascular smooth muscle cells (VSMCs). (A,B) EDU incorporation after culture with 10% FBS at different time points (0, 12, 24, 48 and 96 h). EDU indicated proliferating vascular smooth muscle cells; DAPI showed the total number of cells. ***P<0.001 compared with others. (C,D) miR-30e and miR-1 levels at different time points (0, 12, 24, 48 and 96 h) during active proliferation following 10% FBS stimulation. ***P<0.001 vs. 0, 12, 48 and 96 h. miR-1 had no change. (E,F) Platelet-derived growth factor-BB (PDGF-BB; 20 ng/mL) caused a time-dependent increase in Transwell culture. ***P<0.001 compared with control (CTR) incubated. (a–f) stimulated with PDGF-BB 0, 1, 2, 4, 6 and 8 h; (g–l) stimulated with CTR. (G,H) PDGF-BB caused miR-30e a time-dependent increase. ***P<0.001 vs. 0 h; miR-1 had no change. Data are expressed as the mean±SD (n=3).

MiR-30e Regulation of VSMC Migration In Vitro

To confirm whether miR-30e also influences VSMC migration, we treated VSMCs with PDGF-BB at 0, 1, 2, 4, 6 and 8 h. PDGF-BB caused a time-dependent increase in VSMC migration (Figure 2E,F). To investigate the potential link between miR-30e, miR-1 and VSMCs migration, the expression of miR-30e and miR-1 was determined in non-migrating and migrating VSMCs; the latter being stimulated by PDGF-BB. As shown in Figure 3, the expression of miR-30e after 8 h in PDGF-BB-treated VSMCs was significantly lower than at 0 h, and the change was time dependent (Figure 2G). In contrast, miR-1 levels remained unchanged (Figure 2H). These results demonstrated that the expression of miR-30e was modulated in migrating VSMCs.

Figure 3.

Knockdown of miR-30e increased proliferation and migration of vascular smooth muscle cells (VSMCs) in vivo and in vitro. (A) miR-30e mimic increases miR-30e levels in a dose-dependent manner. ***P<0.001 vs. 0 nmol/L. (B) miR-30e inhibitor decline miR-30e levels in a dose-dependent manner. ***P<0.001 vs. 0 nmol/L and 100 nmol/L. (C) miR-30e overexpression by mimic transfection and reduced by inhibitor transfection. ***P<0.001 vs. control (CTR). **P<0.01 vs. CTR. (D) miR-1 overexpression by mimic transfection and reduced by inhibitor transfection. ***P<0.001 vs. CTR, **P<0.01 vs. CTR. (E,F) EDU incorporation to detect the proliferation. miR-30e mimic transfection reduced the proliferation of VSMCs. miR-1 has no changed. **P<0.01 vs. CTR. (G) Transwell to detect the proliferation. miR-30e mimic transfection reduced the migration of VSMCs. (I–K) The protein expression levels of PCNA indicated the proliferation in vivo, and the protein expression levels of ICAM-1 showed migration in vivo. *P<0.05 vs. control (CTR). Data are expressed as the mean±SD (n=3).

Knocking Down of miR-30e Increases the Proliferation and Migration In Vitro and In Vivo

We performed loss- and gain-of-function studies using miRNA inhibitors or mimics to knock down or overexpress miR-30e and miR-1 in VSMCs. We determined the effect of transfection by using RT-PCR. As shown in Figure 3, transfection of VSMCs with the mimic resulted in a dose-dependent increase in expression (Figure 3A), whereas transfection with the inhibitor resulted in a dose-dependent decline (Figure 3B). To confirm the specific role of miR-30e in proliferation and migration, VSMCs were transfected with miR-30e and miR-1 mimic, miR-30e and miR-1 inhibitor, or negative controls. As shown in Figure 3, transfection with the miR-30e inhibitor caused an increase in proliferation (Figure 3E) and migration (Figure 3G), while the miR-30e mimic reduced these processes. miR-30e inhibited PDGF-BB- or serum-induced VSMC proliferation and migration, as determined by Edu incorporation and Transwell assays. In contrast, the miR-1 mimic and inhibitor had no effects (Figure 3F,H). Western blot showed that overexpression of miR-30e decreased the protein level of PCNA and ICAM-1 compared with the control group in vivo (P<0.01). Downregulated miR-30e increased the protein level of PCNA and ICAM-1 compared with the miR-30e group in vivo (P<0.01). There were no differences between the control group and the miR-30e scramble group (P>0.05) (Figure 3I–K).

MiR-30e Regulation of VSMC Differentiation

VSMC differentiation is accompanied by decreased proliferation and migration. The different phenotypic switches were marked with α-SMA. We also found that the phenotypic switch of VSMCs marked with α-SMA was decreased in de-differentiated VSMCs transfected with the miR-30e inhibitor (Figure 4A,B). VSMC differentiation was increased when migration decreased. Compared with the CTR group, mRNA levels of ACTA2 was decreased while mRNA levels of MYH11 was increased, and miR-30e mimic reversed these changes after incubation with PDGF-BB in the miR-CTR group (Figure 4C,D).

Figure 4.

Knockdown miR-30e reduced the phenotypic switch of vascular smooth muscle cells (VSMCs). (A) The reduced expression of α-SMA in VSMCs incubated with 10% FBS indicated the phenotypic switch of VSMCs. (B) The reduced expression of α-SMA in VSMCs incubated with platelet-derived growth factor-BB (PDGF-BB) indicated the phenotypic switch of VSMCs. (C,D) The mRNA level of ACTA2 was downregulated, but MYH11 was upregulated after stimulation of PDGF-BB; these changes were reversed by miR-30e mimic. *P<0.05, ***P<0.001 vs. Control (CTR); ^##P<0.01, ^###P<0.001 vs. miR-CTR; ^&&&P<0.001 vs. miR-30e mimic. Data are expressed as the mean±SD (n=3).

Identification of the Target Gene of MiR-30e in VSMCs

In order to identify the targets of miR-30e, we used TargetScan and identified Ube2i as a potential target for regulating VSMC proliferation and migration in vitro; it is upregulated in animal models of vascular injury. We built reporter plasmids containing potential miRNA binding sites of rat Ube2i at the position of the luciferase reporter gene (wt-Ube2i) or containing a mutated sequence of the miRNA binding site (mut-Ube2i); these were transfected into VSMCs stably overexpressing miR-30e or miR-1. wt-Ube2i with overexpressing miR-30e sequences displayed significantly lower luciferase activity than the empty vector or their respective mutated constructs (Figure 5A), indicating the functionality of putative Ube2i 3’UTR miR-30e seed sequences. Ube2i protein was upregulated in balloon-injured rat carotid arteries from 0 to 14 d (Figure 5B,C). In quiescent and proliferating VSMCs, Ube2i mRNA and protein were maximally enriched at the 24-h time point (Figure 5D,H). While VSMCs were stimulated for migration, Ube2i mRNA and protein levels were maximal at the 8-h time point (Figure 5E,I). The mRNA level of Ube2i was higher than in controls after miR-30e inhibitor transfection, and lower after miR-30e mimic transfection (Figure 5F,J). Western blotting demonstrated that inhibition of miR-30e increased, and overexpression of miR-30e decreased Ube2i protein levels in relation to controls in vitro and in vivo (Figure 5G,K,L). Changes in Ube2i mRNA and protein levels followed a trend opposite to that of miR-30e.

Figure 5.

Knockdown miR-30e increased the ubiquitin-conjugating enzyme E2I (Ube2i) levels in vivo and in vitro. (A) Luciferase assay of the wild-type (WT) and mutant (Mut) Ube2i 3’untranslated region (UTR) in miR-30e mimic-, miR-30e inhibitor-, miR-1 mimic- and miR-1 inhibitor-treated vascular smooth muscle cells (VSMCs). ***P<0.001 vs. other groups. (B) Representative hematoxylin-eosin (H-E) stained photomicrographs of the vascular walls of rat carotid arteries at different time points after balloon injury: 0 days (a), 3 days (b), 7 days (c) and 14 days (d). (C) The Ube2i protein was upregulated in balloon-injured rat carotid arteries from 0 d to 14 d. (D,H) Ube2i mRNA and protein levels at 0, 12, 24, 48, and 96 h during active proliferation following 10% FBS stimulation. ***P<0.001 vs. 0, 12, 24, 48 and 96 h. *P<0.05 vs. 0, 12, 48 and 96 h. (E,I) Platelet-derived growth factor-BB (PDGF-BB) caused Ube2i a time-dependent reduce at mRNA and protein levels. ***P<0.001 vs. 0 h. (F,J) miR-30e mimic reduced Ube2i mRNA levels, and miR-30e inhibitor increased Ube2i mRNA levels, after stimulation by 10% FBS. miR-1 caused no change. ***P<0.001 vs. others. (G,K) miR-30e mimic reduced Ube2i protein levels, and miR-30e inhibitor increased Ube2i protein levels, after stimulation by PDGF-BB. miR-1 caused no change. (L) overexpression of miR-30e reduced Ube2i protein levels, and reduction of miR-30e increased Ube2i protein levels in the balloon-injured carotid artery. ***P<0.001 vs. others. Data are expressed as the mean±SD (n=3).

Knockdown of Ube2i Downregulated the Proliferation and Migration of VSMCs With Low NF-κB Levels

The siRNA-mediated knockdown reduced Ube2i mRNA expression to below the level of controls (Figure 6A). As the results revealed, proliferation and migration of VSMCs, as detected by Edu dyeing and Transwell chambers after stimulation with 10% serum and 20ng/mL PDGF-BB, were downregulated after knock-down of Ube2i (Figure 6B,C).

Figure 6.

Ubiquitin-conjugating enzyme E2I (Ube2i) regulates the proliferation and migration of cultured vascular smooth muscle cells (VSMCs). (A) Knockdown Ube2i mRNA levels. (B) The effect of Ube2i on the proliferation of cultured VSMCs. (C) The effect of Ube2i on the migration of cultured VSMCs. ***P<0.001 vs. control (CTR). Data are expressed as the mean±SD (n=3).

The protein levels of IκBα and P65 were different after miR-30e mimic transfection in cells stimulated by PDGB-BB (20 ng/mL) for 8 h. The protein levels of IκBα and P65 were lower after miR-30e transfection (Figure 7A–C); this was also detected in the groups that had a knockdown of Ube2i (Figure 7D–F).

Figure 7.

Downregulated proliferation and migration of vascular smooth muscle cells (VSMCs) accompanied low NF-κB levels. (A–C) miR-30e mimic transfection reduced IKBα and P65 protein levels after platelet-derived growth factor-BB (PDGF-BB) stimulation. ***P<0.001 vs. others; ^###P<0.001 vs. others; ^&&&P<0.001 vs. others. (D–F) Knockdown Ube2i reduced IKBα and P65 protein levels after PDGF-BB stimulation. ***P<0.001 vs. others; ^###P<0.001 vs. others; ^&&&P<0.001 vs. others. Data are expressed as the mean±SD (n=3).

Knockdown of MiR-30e Inhibits Apoptosis in Cultured VSMCs In Vitro and In Vivo

The apoptosis-related proteins were tested by Western blotting, and the rate of cell apoptosis was detected by Annexin-V APC/PI double staining flow cytometry. The apoptosis-related proteins did not alter after incubation with PDGF-BB in the miR-CTR (negative control) group. Both the Bcl-2 and Bax expression levels were downregulated and upregulated, respectively, in the miR-30e mimic group compared with the CTR group. However, Bcl-2 and Bax expression levels were reversed in the miR-30e inhibitor group. The level of cleaved caspase-3 level was increased in the miR-30e mimic group compared with the CTR group, while it decreased in the miR-30e inhibitor group (Figure 8A–D). Annexin-V APC/PI double staining flow cytometry showed that the apoptotic rate of the miR-30e mimic group was increased, but decreased in the miR-30e inhibitor group compared with the CTR group (Figure 8E,F). Similar effects of miR-30e on apoptosis were also detected in vivo (Figure 8G–I).

Figure 8.

miR-30e regulation in cultured vascular smooth muscle cells (VSMCs) apoptosis in vivo and in vitro. (A–D) Represents apoptosis-related proteins, Bcl-2, Bax and cleaved caspase-3 after platelet-derived growth factor-BB (PDGF-BB) stimulation. (E,F) The apoptosis rate was increased after transfection with the miR-30e mimic, but decreased after transfection with the miR-30e inhibitor. (G–I) Apoptosis-related proteins, Bcl-2, Bax and cleaved caspase-3 in vivo. *P<0.05, **P<0.01, ***P<0.001 vs. the control (CTR); ^###P<0.001 vs. the miR-30e mimic. Data are expressed as the mean±SD (n=3).

Discussion

MiRNAs have been implicated in the development of cardiovascular diseases. Recent studies have demonstrated that miRNAs such as miR-143/145, miR-146a, and miR-221 are critical targets of multiple growth factor signaling pathways that regulate VSMC proliferation and migration.^31–35 The molecular mechanisms underlying the regulation of VSMC proliferation and migration by these miRNAs are divergent, but are mainly involved in the direct or indirect regulation of the expression of target genes such as serum response factor, myocardin and myocardin-related transcription factors, and Kruppel-like zinc finger family proteins.³⁶ For example, miR-221/222 inhibits the expression of its target genes, p27Kip1 and p57Kip2, thus promoting the proliferation and migration processes;³⁷ and miR-143 and miR-145 are highly expressed in VSMCs and help maintain the phenotype and regulate proliferation and migration.³⁸ Our studies provide significant novel insights into the critical importance and functional complexities of miRNAs in regulating VSMCs proliferation, migration and the development of vascular diseases. Previous publications have provided little information about miR-30e regulation of VSMC behavior.

MiR-30e is a member of the miR-30 family with which it shares similar targets: miR-30a/b/c/d/e and miR-384-5p. We observed that miR-30e was the most expressed member in VSMCs and other members of the miR-30 family were not implicated in following the stimulating by PDGF-BB. This study is to illustrate the essential role of miR-30e in VSMC proliferation, migration and apoptosis in vitro, substantiating miR-30e as a novel modulator. We found that the expression level of miR-30e in VSMCs was nearly 1000-fold that of miR-1, which has already been demonstrated experimentally to have only minor effects on VSMC proliferation.^39,40 Therefore, it is necessary to compare miR-30e with other known vascular well-expressed miRNA, such as miR-221/222 (which increases during proliferation). We observed that the expression level of miR-30e was approximately close to that of miR-221, which is abundantly expressed in VSMCs.³²

We demonstrated that miR-30e expression is distinctly downregulated in proliferating and migrating VSMCs and upregulated in quiescent ones. After transfection with a miR-30e mimic, proliferation and migration were reduced and the cell phenotype tended toward differentiation. When a miR-30e inhibitor was transfected, proliferation and migration were increased and the cell phenotype tended toward dedifferentiation. We also found that miR-30e could increase the rate of apoptosis in VSMCs. That means that miR-30e inhibits proliferation and migration as well as promotes apoptosis in VSMCs. It is well known that VSMC proliferation and migration can lead to atherosclerosis;^41,42 our results indicate that miR-30e exerts an anti-atherosclerosis effect via inhibiting proliferation and migration and promoting the apoptosis of VSMCs. Interestingly, we also found that overexpression of miR-30e markedly increased the expression of VSMC differentiation markers such as α-SMA, suggesting that miR-30e could also be critical for VSMC differentiation. Both loss-of-function and gain-of-function studies suggest that miR-30e plays a key role in VSMCs behavior in vitro. Furthermore, in order to demonstrate that miR-30e inhibits VSMCs proliferation and migration.

The miRNAs have been recognized to mediate PDGF-induced cell dysregulation; their exact functions remain to be elucidated. But mechanisms of some miRNAs were elucidated recently. For example, induction of miRNAs-146b by PDGF-BB is modulated via MAPK-dependent induction of c-fos.⁴³

MicroRNA-328 was found as a prominent downregulated microRNA, displaying a specific dose- and time-dependent downregulation by PDGF-BB treatment⁴⁴ in pulmonary artery smooth muscle cells in which proliferation and migration were inhibited by microRNA-328. The decrease of microRNA-328 by PDGF-BB might be due to the increased expression of DNA methylation transferase 1 and DNA methylation.⁴⁴ We speculated that miR-30e may involve in the similar mechanism by reduction of PDGF-BB.

Recently, Liu et al⁴⁵ reported that miR-30c and miR-30e inhibits neointimal hyperplasia by targeting Ca²⁺/ calmodulin-dependent protein kinase IIδ (CaMKIIδ). While one miRNA may have several target mRNAs, while one mRNA may have several miRNA regulators. We searched for evolutionarily conserved targets of miR-30e by TargetScan. After transient cotransfection with WT-Ube2i-3’UTR and miR-30e mimic into cultured VSMCs, fluorescence was reduced. The luciferase reporter assay result proved that Ube2i is a direct target for miR-30e. Our study indicated that miR-30e is an important regulator of Ube2i expression, and Ube2i is a direct target for this miRNA. Meanwhile, we found that the trends of Ube2i levels were diametrically opposite to those of miR-30e, including in inhibiting VSMC proliferation and migration.

Ube2i modifies key enzymes in the inhibition of target protein degradation; maintaining and adjusting target protein levels are important for many processes. The first SUMO-modified protein implicated in NF-κB regulation is IκBα.⁴⁶ This NF-κB inhibitor plays multiple critical roles in regulating both the initial activation of NF-κB and the duration of this activity in response to extracellular signals. Ube2i interacts with IκBα, and overexpression of a catalytically inactive mutant of Ube2i delays both IκBα degradation and NF-κB activation.⁴⁷ However, in the regulation of VSMC proliferation and migration, its specific mechanism needs further research. In our study, balloon-injured vessels expressed different Ube2i levels at different days; expression was higher on later days. The thicker the neointima, the greater its content. Downregulation of Ube2i in VSMCs reduced the IκBα and NFκB levels. We found that miR-30e inhibits VSMC proliferation and migration partially through targeting the transcription factor, Ube2i, and downregulating the IκBα/NFκB signal pathway. Ube2i has also been demonstrated to be deregulated in cardiomyocytes, which are transfected with pre-miR-199a.⁴⁸ Hence, Ube2i was identified to be target of another miRNA, which indicated that Ube2i may be modulated by more kinds of miRNA in several cells.

miR-30e expression was substantially downregulated in proliferating and migrating VSMCs, and restoration of its expression markedly inhibited both proliferation and migration. We examined the effect of miR-30e on these transcription factors and found that modulation of miR-30e expression significantly affected the expression of Ube2i, a major regulator of IκBα and NF-κB expression.^49,50 NF-kB is one of the known SUMO targets.⁴⁹ Sumoylation inactivates NF-kB by conjugation with IkB.⁵¹ Carbia-Nagashima et al⁵⁰ reported that RWD-containing sumoylation enhancer (RSUME) increased IkB levels by promoting the sumoylation of IkB, leading to an inhibition of NF-kB transcriptional activity and target expression. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1a, promoting its stabilization and transcriptional activity during hypoxia.

Recently, miR-146a was demonstrated as regulator of the maturation and differentiation of VSMCs via targeting NF-κB gene.⁷ We speculated that VSMCs were modulated by several miRNAs targeting several signaling pathways.

In summary, the present study identifies that miR-30e exerts anti-atherosclerosis effects via inhibiting proliferation and migration and promoting apoptosis in VSMCs. More explicitly, we demonstrate that miR-30e inhibits the proliferation and migration of VSMCs partially by targeting the transcription factor, Ube2i, and downregulating the IκBα/NFκB signaling pathway.

Acknowledgments

This work was supported by the Natural Science Foundation of China (81570326). Y.Z. and D.Y. designed the study and analysed and interpreted data. Y.Z., P.W., C.N., F.H., W.G. and N.Z. performed the experiments. Y.L. and T.X. collected data. Y.Z., C.N. and P.W. analysed results and performed statistical analysis. Y.Z., C.N., P.W. and D.Y. wrote the manuscript. All authors read and approved the final manuscript.

Disclosures

None.

References

• 1.   Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature 2011; 473: 317–325.
• 2.   Robinson JG, Gidding SS. Curing atherosclerosis should be the next major cardiovascular prevention goal. J Am Coll Cardiol 2014; 63: 2779–2785.
• 3.   Rensen SS, Doevendans PA, van Eys GJ. Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Neth Heart J 2007; 15: 100–108.
• 4.   Li L, Zhang HN, Chen HZ, Gao P, Zhu LH, Li HL, et al. SIRT1 acts as a modulator of neointima formation following vascular injury in mice. Circ Res 2011; 108: 1180–1189.
• 5.   Song MC, Kim EC, Kim WJ, Kim TJ. Meso-dihydroguaiaretic acid inhibits rat aortic vascular smooth muscle cell proliferation by suppressing phosphorylation of platelet-derived growth factor receptor beta. Eur J Pharmacol 2014; 744: 36–41.
• 6.   Bayes-Genis A, Conover CA, Schwartz RS. The insulin-like growth factor axis: A review of atherosclerosis and restenosis. Circ Res 2000; 86: 125–130.
• 7.   Dong S, Xiong W, Yuan J, Li J, Liu J, Xu X. MiRNA-146a regulates the maturation and differentiation of vascular smooth muscle cells by targeting NF-kappaB expression. Mol Med Rep 2013; 8: 407–412.
• 8.   Merlet E, Atassi F, Motiani RK, Mougenot N, Jacquet A, Nadaud S, et al. miR-424/322 regulates vascular smooth muscle cell phenotype and neointimal formation in the rat. Cardiovasc Res 2013; 98: 458–468.
• 9.   Wang Z, Zhang D, Hu Z, Cheng J, Zhuo C, Fang X, et al. MicroRNA-26a-modified adipose-derived stem cells incorporated with a porous hydroxyapatite scaffold improve the repair of bone defects. Mol Med Rep 2015; 12: 3345–3350.
• 10.   Liu C, Tang X. Downregulation of microRNA-210 inhibits osteosarcoma growth in vitro and in vivo. Mol Med Rep 2015; 12: 3674–3680.
• 11.   Schober A, Nazari-Jahantigh M, Wei Y, Bidzhekov K, Gremse F, Grommes J, et al. MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1. Nat Med 2014; 20: 368–376.
• 12.   Wang WP, Ho PY, Chen QX, Addepalli B, Limbach PA, Li MM, et al. Bioengineering novel chimeric microRNA-34a for prodrug cancer therapy: High-yield expression and purification, and structural and functional characterization. J Pharmacol Exp Ther 2015; 354: 131–141.
• 13.   Wei C, Song H, Sun X, Li D, Song J, Hua K, et al. miR-183 regulates biological behavior in papillary thyroid carcinoma by targeting the programmed cell death 4. Oncology Reports 2015; 34: 211–220.
• 14.   Small EM, Frost RJ, Olson EN. MicroRNAs add a new dimension to cardiovascular disease. Circulation 2010; 121: 1022–1032.
• 15.   Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, Hammond SM, et al. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet 2006; 38: 228–233.
• 16.   Kontaraki JE, Marketou ME, Zacharis EA, Parthenakis FI, Vardas PE. Differential expression of vascular smooth muscle-modulating microRNAs in human peripheral blood mononuclear cells: Novel targets in essential hypertension. J Hum Hypertens 2014; 28: 510–516.
• 17.   Markou A, Sourvinou I, Vorkas PA, Yousef GM, Lianidou E. Clinical evaluation of microRNA expression profiling in non small cell lung cancer. Lung Cancer 2013; 81: 388–396.
• 18.   Wu F, Zhu S, Ding Y, Beck WT, Mo YY. MicroRNA-mediated regulation of Ubc9 expression in cancer cells. Clin Cancer Res 2009; 15: 1550–1557.
• 19.   Wang Y, Li L, Qu Z, Li R, Bi T, Jiang J, et al. The expression of miR-30a* and miR-30e* is associated with a dualistic model for grading ovarian papillary serious carcinoma. Int J Oncol 2014; 44: 1904–1914.
• 20.   Schepeler T, Holm A, Halvey P, Nordentoft I, Lamy P, Riising EM, et al. Attenuation of the beta-catenin/TCF4 complex in colorectal cancer cells induces several growth-suppressive microRNAs that target cancer promoting genes. Oncogene 2012; 31: 2750–2760.
• 21.   Presneau N, Eskandarpour M, Shemais T, Henderson S, Halai D, Tirabosco R, et al. MicroRNA profiling of peripheral nerve sheath tumours identifies miR-29c as a tumour suppressor gene involved in tumour progression. Br J Cancer 2013; 108: 964–972.
• 22.   Hu Y, Correa AM, Hoque A, Guan B, Ye F, Huang J, et al. Prognostic significance of differentially expressed miRNAs in esophageal cancer. Int J Cancer 2011; 128: 132–143.
• 23.   Silva J, Garcia V, Zaballos A, Provencio M, Lombardia L, Almonacid L, et al. Vesicle-related microRNAs in plasma of nonsmall cell lung cancer patients and correlation with survival. Eur Respir J 2011; 37: 617–623.
• 24.   Shen Z, Pardington-Purtymun PE, Comeaux JC, Moyzis RK, Chen DJ. Associations of UBE2I with RAD52, UBL1, p53, and RAD51 proteins in a yeast two-hybrid system. Genomics 1996; 37: 183–186.
• 25.   Wang CM, Brennan VC, Gutierrez NM, Wang X, Wang L, Yang WH. SUMOylation of ATF3 alters its transcriptional activity on regulation of TP53 gene. J Cell Biochem 2013; 114: 589–598.
• 26.   Neyret-Kahn H, Benhamed M, Ye T, Le Gras S, Cossec JC, Lapaquette P, et al. Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation. Genome Res 2013; 23: 1563–1579.
• 27.   Ihara M, Stein P, Schultz RM. UBE2I (UBC9), a SUMO-conjugating enzyme, localizes to nuclear speckles and stimulates transcription in mouse oocytes. Biol Reprod 2008; 79: 906–913.
• 28.   Tahmasebi S, Ghorbani M, Savage P, Gocevski G, Yang XJ. The SUMO conjugating enzyme Ubc9 is required for inducing and maintaining stem cell pluripotency. Stem Cells 2014; 32: 1012–1020.
• 29.   Guo J, Li D, Bai S, Xu T, Zhou Z, Zhang Y. Detecting DNA synthesis of neointimal formation after catheter balloon injury in GK and in Wistar rats: Using 5-ethynyl-2’-deoxyuridine. Cardiovasc Diabetol 2012; 11: 150.
• 30.   Zhang XW, Wang Y, Cai JB, Hou XF, Wang HG, Zou JG, et al. Downregulation of quinone reductase 2 attenuates vascular smooth muscle cells proliferation and neointimal formation in balloon injured rat carotid artery. Cell Physiol Biochem 2012; 29: 453–462.
• 31.   Li P, Liu Y, Yi B, Wang G, You X, Zhao X, et al. MicroRNA-638 is highly expressed in human vascular smooth muscle cells and inhibits PDGF-BB-induced cell proliferation and migration through targeting orphan nuclear receptor NOR1. Cardiovasc Res 2013; 99: 185–193.
• 32.   Liu X, Cheng Y, Yang J, Xu L, Zhang C. Cell-specific effects of miR-221/222 in vessels: Molecular mechanism and therapeutic application. J Mol Cell Cardiol 2012; 52: 245–255.
• 33.   Anand S, Majeti BK, Acevedo LM, Murphy EA, Mukthavaram R, Scheppke L, et al. MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis. Nat Med 2010; 16: 909–914.
• 34.   Boettger T, Beetz N, Kostin S, Schneider J, Kruger M, Hein L, et al. Acquisition of the contractile phenotype by murine arterial smooth muscle cells depends on the Mir143/145 gene cluster. J Clin Invest 2009; 119: 2634–2647.
• 35.   Newman MA, Hammond SM. Emerging paradigms of regulated microRNA processing. Genes Dev 2010; 24: 1086–1092.
• 36.   Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, et al. The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol 2009; 11: 1487–1495.
• 37.   Liu X, Cheng Y, Zhang S, Lin Y, Yang J, Zhang C. A necessary role of miR-221 and miR-222 in vascular smooth muscle cell proliferation and neointimal hyperplasia. Circ Res 2009; 104: 476–487.
• 38.   Hutcheson R, Terry R, Chaplin J, Smith E, Musiyenko A, Russell JC, et al. MicroRNA-145 restores contractile vascular smooth muscle phenotype and coronary collateral growth in the metabolic syndrome. Arterioscler Thromb Vasc Biol 2013; 33: 727–736.
• 39.   Kontaraki JE, Marketou ME, Zacharis EA, Parthenakis FI, Vardas PE. Differential expression of vascular smooth muscle-modulating microRNAs in human peripheral blood mononuclear cells: Novel targets in essential hypertension. J Hum Hypertens 2014; 28: 510–516.
• 40.   Torella D, Iaconetti C, Catalucci D, Ellison GM, Leone A, Waring CD, et al. MicroRNA-133 controls vascular smooth muscle cell phenotypic switch in vitro and vascular remodeling in vivo. Circ Res 2011; 109: 880–893.
• 41.   Zhang T, Zhang X, Yu W, Chen J, Li Q, Jiao Y, et al. Effects of chemokine-like factor 1 on vascular smooth muscle cell migration and proliferation in vascular inflammation. Atherosclerosis 2013; 226: 49–57.
• 42.   Lightell DJ Jr, Moss SC, Woods TC. Loss of canonical insulin signaling accelerates vascular smooth muscle cell proliferation and migration through changes in p27Kip1 regulation. Endocrinology 2011; 152: 651–658.
• 43.   Shao M, Rossi S, Chelladurai B, Shimizu M, Ntukogu O, Ivan M, et al. PDGF induced microRNA alterations in cancer cells. Nucleic Acids Res 2011; 39: 4035–4047.
• 44.   Qian Z, Zhang L, Chen J, Li Y, Kang K, Qu J, et al. MiR-328 targeting PIM-1 inhibits proliferation and migration of pulmonary arterial smooth muscle cells in PDGFBB signaling pathway. Oncotarget 2016; 7: 54998–55011.
• 45.   Liu YF, Spinelli A, Sun LY, Jiang M, Singer DV, Ginnan R, et al. MicroRNA-30 inhibits neointimal hyperplasia by targeting Ca(2+)/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta). Sci Rep 2016; 6: 26166.
• 46.   Desterro JM, Rodriguez MS, Hay RT. SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Mol Cell 1998; 2: 233–239.
• 47.   Tashiro K, Pando MP, Kanegae Y, Wamsley PM, Inoue S, Verma IM. Direct involvement of the ubiquitin-conjugating enzyme Ubc9/Hus5 in the degradation of IkappaBalpha. Proc Natl Acad Sci U S A 1997; 94: 7862–7867.
• 48.   Haghikia A, Missol-Kolka E, Tsikas D, Venturini L, Brundiers S, Castoldi M, et al. Signal transducer and activator of transcription 3-mediated regulation of miR-199a-5p links cardiomyocyte and endothelial cell function in the heart: A key role for ubiquitin-conjugating enzymes. Eur Heart J 2011; 32: 1287–1297.
• 49.   Desterro J, Rodriguez M, Hay RT. SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Molecular Cell 1998; 2: 233–239.
• 50.   Carbia-Nagashima A, Gerez J, Perez-Castro C, Paez-Pereda M, Silberstein S, Stalla GK, et al. RSUME, a small RWD-containing protein, enhances SUMO conjugation and stabilizes HIF-1alpha during hypoxia. Cell 2007; 131: 309–323.
• 51.   Mabb AM, Miyamoto S. SUMO and NF-kappaB ties. Cellular & Molecular Life Sciences Cmls 2007; 64: 1979–1996.