c-Ski inhibits the proliferation of vascular smooth muscle cells via suppressing Smad3 signaling but stimulating p38 pathway

Proliferation of vascular smooth muscle cells (VSMCs) plays key roles in the progression of intimal hyperplasia, but the molecular mechanisms that trigger VSMC proliferation after vascular injury remain unclear. c-Ski, a co-repressor of transforming growth factor β (TGF-β)/Smad signaling, was detected to express in VSMC of rat artery. During the course of arterial VSMC proliferation induced by balloon injury in rat, the endogenous protein expressions of c-Ski decreased markedly in a time-dependent manner. In vivo c-Ski gene delivery was found to significantly suppress balloon injury-induced VSMC proliferation and neointima formation. Further investigation in A10 rat aortic smooth muscle cells demonstrated that overexpression of c-Ski gene inhibited TGF-β1 (1 ng/ml)-induced A10 cell proliferation while knockdown of c-Ski by RNAi enhanced the stimulatory effect of TGF-β1 on A10 cell growth. Western blot for signaling detection showed that suppression of Smad3 phosphorylation while stimulating p38 signaling associated with upregulation of cyclin-dependent kinase inhibitors p21 and p27 was responsible for the inhibitory effect of c-Ski on TGF-β1-induced VSMC proliferation. These data suggest that the decrease of endogenous c-Ski expression is implicated in the progression of VSMC proliferation after arterial injury and c-Ski administration represents a promising role for treating intimal hyperplasia via inhibiting the proliferation of VSMC.     

miR-181c-5p在颅内动脉瘤中通过靶向PTPN4调节血管平滑肌细胞的机制研究

摘要 目的：探讨miR-181c-5p在颅内动脉瘤血管平滑肌细胞（VSMC）表型调节中的生物学功能及其潜在的调控机制。方法：采用实时荧光定量聚合酶链式反应（RT-qPCR）检测miR-181c-5p mRNA在颅内动脉瘤（IA）患者血清中的表达水平。 采用药物细胞毒性实验（CCK8）、集落形成、transwell迁移和流式细胞仪检测过表达miR-181c-5p介导的VSMC细胞表型的变化。采用双荧光素酶报告基因检测miR-181c-5p的潜在靶标。结果：IA患者血清中的miR-181c-5p表达水平高于健康体检者（P<0.05）。miR-181c-5p的过表达显着抑制了VSMC增殖、克隆形成和迁移,同时刺激了细胞凋亡（P<0.05）。PTPN4被证实是miR-181c-5p的直接靶标,而miR-181c-5p的过表达导致PTPN4在VSMC中低表达。结论：miR-181c-5p / PTPN4介导的VSMC表型调节可能部分导致IA病变。     

Augmentation of miR-202 in varicose veins modulates phenotypic transition of vascular smooth muscle cells by targeting proliferator–activated receptor-γ coactivator-1α

In varicose veins, vascular smooth muscle cells (VSMCs) often show abnormal proliferative and migratory rates and phenotypic transition. This study aimed to investigate whether microRNA (miR)-202 and its potential target, peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α), were involved in VSMC phenotypic transition. miR-202 expression was analyzed in varicose veins and in VSMCs conditioned with platelet-derived growth factor. The effect of miR-202 on cell proliferation and migration was assessed. Furthermore, contractile marker SM-22α, synthetic markers vimentin and collagen I, and PGC-1α were analyzed by Western blot analysis. The modulation of PGC-1α expression by miR-202 was also evaluated. In varicose veins and proliferative VSMCs, miR-202 expression was upregulated, with decreased SM-22α expression and increased vimentin and collagen I expression. Transfection with a miR-202 mimic induced VSMC proliferation and migration, whereas a miR-202 inhibitor reduced cell proliferation and migration. miR-202 mimic constrained luciferase activity in HEK293 cells that were cotransfected with the PGC-1α 3′-untranslated region (3′-UTR) but not those with mutated 3′-UTR. miR-202 suppressed PGC-1α protein expression, with no influence on its messenger RNA expression. PGC-1α mediated VSMC phenotypic transition and was correlated with reactive oxygen species production. In conclusion, miR-202 affects VSMC phenotypic transition by targeting PGC-1α expression, providing a novel target for varicose vein therapy.     

MiR-34a/miR-93 target c-Ski to modulate the proliferaton of rat cardiac fibroblasts and extracellular matrix deposition in vivo and in vitro

Cardiac fibrosis is associated with diverse heart diseases. In response to different pathological irritants, cardiac fibroblasts may be induced to proliferate and differentiate into cardiac myofibroblasts, thus contributing to cardiac fibrosis. TGF-β signaling is implicated in the development of heart failure through the induction of cardiac fibrosis. C-Ski, an inhibitory regulator of TGF-β signaling, has been reported to suppress TGF-β1-induced human cardiac fibroblasts' proliferation and ECM protein increase; however, the underlying molecular mechanism needs further investigation. In the present study, we demonstrated that c-Ski could ameliorate isoproterenol (ISO)-induced rat myocardial fibrosis model and TGF-β1-induced primary rat cardiac fibroblasts' proliferation, as well as extracellular matrix (ECM) deposition. The protein level of c-Ski was dramatically decreased in cardiac fibrosis and TGF-β1-stimulated primary rat cardiac fibroblasts. In recent decades, a family of small non-coding RNA, namely miRNAs, has been reported to regulate gene expression by interacting with diverse mRNAs and inducing either translational suppression or mRNA degradation. Herein, we selected miR-34a and miR-93 as candidate miRNAs that might target to regulate c-Ski expression. After confirming that miR-34a/miR-93 targeted c-Ski to inhibit its expression, we also revealed that miR-34a/miR-93 affected TGF-β1-induced fibroblasts' proliferation and ECM deposition through c-Ski. Taken together, we demonstrated a miR-34a/miR-93-c-Ski axis which modulates TGF-β1- and ISO-induced cardiac fibrosis in vitro and in vivo; targeting the inhibitory factors of c-Ski to rescue its expression may be a promising strategy for the treatment of cardiac fibrosis.     

MicroRNA-30c contributes to the development of hypoxia pulmonary hypertension by inhibiting platelet-derived growth factor receptor β expression

Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs). MicroRNAs have been implicated in the regulation of cell proliferation and might be implicated in the etiology of PAH. Data from in vivo and in vitro cell culture models showed that hypoxia inhibits microRNA-30c (miR-30c) expression in PASMCs. Inhibition of miR-30c by either hypoxia or AMO-30c results in PASMC proliferation (cell viability, 5-bromo-2-deoxyuridine (BrdU) incorporation, proliferating cell nuclear antigen, Ki67, and tubulin polymerization) and the inhibition of apoptosis (cell cycle progression, Cyclin A and Cyclin D, and TUNEL staining). Moreover, down-regulation of miR-30c also results in the phenotype switch from contractile to synthetic PASMC (SM22α and Calponin, osteopontin expression, and wound healing assay). In contrast, these effects were reversed by the application of an miR-30c mimetic under hypoxic conditions. Mechanically, miR-30c inhibited the platelet-derived growth factor receptor β (PDGFRβ) expression by directly binding to the 3′ untranslated region of PDGFRβ mRNA (luciferase reporter assays, and PDGFRβ-masking antisense oligodeoxynucleotides). Pharmacological inhibition of PDGFR by AG-1296 displayed similar effects to the miR-30c mimetic. These data suggest that the down-regulation of miR-30c accounts for the up-regulation of PDGFRβ expression, and subsequent activation of PDGF signaling results in the hypoxia-induced PASMC proliferation and phenotype switching. Therefore, increasing miR-30c expression levels could be explored as a potential new therapy for hypoxia-induced PAH.     

Regulation of vascular smooth muscle cell proliferation, migration and death by heparan sulfate 6-O-endosulfatase1

Migration, proliferation and death of vascular smooth muscle cells (VSMC) are important events in vascular pathology regulated by heparan sulfate proteoglycans and hence potentially by cell surface HS 6-O-endosulfatase1 (sulf1). Sulf1 mRNA expression was increased in cultured VSMC compared to rat aorta. Furthermore, adenovirus mediated overexpression of quail sulf1 decreased adhesion, and increased proliferation and apoptosis of VSMC. Overexpression of a dominant negative variant also decreased adhesion of VSMC and increased proliferation, apoptosis, migration and chemotaxis of VSMC. Our results imply that only normal levels of 6-O-sulfation maintained by sulf1 are optimal for several functions of VSMC.     

miR-103a-2-5p/miR-30c-1-3p inhibits the progression of prostate cancer resistance to androgen ablation therapy via targeting androgen receptor variant 7

Androgens and androgen receptors are vital factors involved in prostate cancer progression, and androgen ablation therapies are commonly used to treat advanced prostate cancer. However, the acquisition of androgen ablation therapy resistance remains a challenge. Recently, androgen receptor splicing variants lacking the ligand-binding domain have been reported to play a critical role in the acquisition of androgen ablation therapy resistance. In the present study, we revealed that the messenger RNA expression and the protein levels of an androgen receptor variant 7 (AR-V7) were higher in prostate cancer tissue samples and in the AR-positive prostate cancer cell line, VCaP. In contrast, microRNA (miR)-30c-1-3p/miR-103a-2-5p expression was significantly downregulated in tumor tissues and cells. miR-30c-1-3p/miR-103a-2-5p overexpression could inhibit AR-V7 expression, suppress VCaP cell growth, and inhibit AR-V7 downstream factor expression by directly targeting the 3′-untranslated region of AR-V7. Under enzalutamide (Enza) treatment, the effects of AR-V7 overexpression were the opposite of those of miR-103a-2-5p/miR-30c-1-3p overexpression; more importantly, the effects of miR-103a-2-5p/miR-30c-1-3p overexpression could be significantly reversed by AR-V7 overexpression under Enza. In summary, we demonstrated a novel mechanism of the miR-30c-1-3p/miR-103a-2-5p/AR-V7 axis modulating the cell proliferation of AR-positive prostate cancer cells via AR downstream targets. The clinical application of miR-30c-1-3p/miR-103a-2-5p needs further in vivo validation.     

miR-92a inhibits vascular smooth muscle cell apoptosis: role of the MKK4–JNK pathway

Vascular smooth muscle cell (VSMC) apoptosis plays an important role in vascular remodeling and atherosclerotic plaque instability. Oxidative stress in diseased vessels promotes VSMC apoptosis in part by activating the c-Jun N-terminal kinase (JNK) pathway, which has been identified as a molecular target of miR-92a in macrophages. Here, we examined the expression and biological activity of miR-92a in VSMC. Quiescent VSMC exhibited a low basal expression of miR-92a, which was positively regulated by serum stimulation and negatively regulated by H₂O₂. Overexpression of miR-92a decreased H₂O₂-induced VSMC apoptosis as indicated by TUNEL assay and cleaved caspase-3 protein levels. Using 3′UTR-reporter assay, we found that miR-92a overexpression led to suppression of both mitogen-activated protein kinase kinase 4 (MKK4)- and JNK1-dependent luciferase activity. We also found that 10 mer seed match between miRNA:mRNA pair is more efficient than 8 mer seed match for us to identify authentic miRNA target. Protein levels of active phospho-JNK and phospho-c-Jun, downstream targets of the MKK4–JNK1 pathway, were also decreased by overexpressing miR-92a in VSMC under oxidative stress. Consistent with these findings, overexpression of MKK4 reversed the anti-apoptotic effects of miR-92a in oxidatively stressed VSMC. In conclusion, miR-92a overexpression inhibits H₂O₂-induced VSMC apoptosis by directly targeting the MKK4–JNK1 pathway.     

PPARγ up-regulates TGFβ/smad signal pathway repressor c-Ski

TGFβ/smad pathway is recognized as an important signal pathway to promote the pathogenesis of atherosclerosis (AS). Peroxisome proliferator-activated receptor γ (PPARγ) activation is considered to be important in modulating AS. Herein, we investigated the regulation of PPARγ on c-Ski, the repressor of TGFβ/smad pathway, in rat AS model and cultured vascular smooth muscle cells (VSMCs). c-Ski mRNA and protein expression were detected by real-time PCR and Western blot, respectively, in vivo and in vitro with treatment of PPARγ agonist rosiglitazone and antagonist GW9662. The proliferation and collagen secretion of VSMCs after c-Ski transfection were investigated. The underlying mechanism was further investigated by online program NUBIScan and luciferase reporter gene analysis. Results showed that both mRNA and protein expressions of c-Ski in the AS lesions was down-regulated in vivo, while in cultured VSMCs, c-Ski transfection significantly suppressed the proliferation and collagen secretion of rat VSMCs. Rosiglitazone significantly up-regulated mRNA and protein levels of c-Ski in VSMCs, which could be blocked by GW9662. Online NUBIScan analysis suggested possible PPARγ binding sites in the promoter region of c-Ski. In addition, luciferase activity of c-Ski reporter gene was also increased obviously in the presence of rosiglitazone. These results indicate that c-Ski is one of the newly found target genes of PPARγ and thus involved in the anti-AS effect of PPARγ.     

Decreased expression of circ_0020397 in intracranial aneurysms may be contributing to decreased vascular smooth muscle cell proliferation via increased expression of miR-138 and subsequent decreased KDR expression

Dysfunction of vascular smooth muscle cells (VSMCs) mediates intracranial aneurysm (IA). KDR is reported to alleviate IA progression via promoting VSMC proliferation, while the upstream regulators are still unclear. Arterial wall tissues at the aneurysm site from 12 patients were obtained. The real-time PCR result indicated that circRNA_0020397 was down-regulated, but miR-138 was up-regulated in artery wall tissues and cells of IA. Overexpressed circRNA_0020397 promoted proliferation of human umbilical artery SMCs. MiR-138 negatively regulated KDR via binding with 3’UTR of KDR mRNA. The expression of circRNA_0020397 was negatively correlated with miR-138. In conclusion, our findings demonstrated that decreased expression of circRNA_0020397 in IA may contribute to the decreased VSMC proliferation via increasing miR-138 expression and subsequently decreasing KDR expression.     

A novel mechanism of Smads/miR-675/TGFβR1 axis modulating the proliferation and remodeling of mouse cardiac fibroblasts

Cardiac fibroblasts (CFs) can over-proliferate during the progression of cardiac fibrosis, accompanied by a net accumulation of extracellular matrix proteins. Based on the profibrotic actions of transforming growth factor beta 1 (TGFβ1), investigating the mechanisms of TGFβ1 function in CFs may provide new directions to treatment for cardiac fibrosis. microRNAs (miRNAs) could control CFs proliferation or remodeling via binding to 3′-untranslated region of messenger RNA (mRNA) to negatively regulate gene expression. In the present study, we downloaded several microarray analyses results from GEO attempting to identify miRNAs and possible downstream targets that may be involved in TGF-β1 function in CFs and to detect the cellular and molecular functions of the identified miRNA–mRNA axis. Here, we identified miR-675 as a downregulated miRNA by TGFβ1 by bioinformatics analyses and experimental verification. Upon TGFβ1 stimulation, the protein levels of Α-SMAΑ-SMA, collagen I, and POSTN, and the secreted collagen in the cell culture supernatant significantly increased, whereas the miR-675 expression decreased. Smads mediate TGFβ1-induced suppression on miR-675 via binding miR-675 promoter region. miR-675 overexpression could inhibit, whereas miR-675 inhibition could enhance TGFβ1-induced mouse CFs (MCF) remodeling and proliferation. TGFβ receptor 1 (TGFβR1), a critical receptor in TGFβ1/Smad signaling, is a direct downstream target of miR-675. TGFβR1 overexpression significantly reverses the effect of miR-675 overexpression on MCF remodeling and proliferation. In summary, miR-675 targets TGFβR1 to attenuate TGFβ1-induced MCF remodeling and proliferation. We demonstrate a novel mechanism of the Smads/miR-675/TGFβR1 axis modulating TGFβ1-induced MCF remodeling and proliferation.     

miR-214-5p targets KLF5 and suppresses proliferation of human hepatocellular carcinoma cells

MicroRNAs (miRNAs) are small endogenous conserved RNAs regulating genes expression through base pairing with the 3′-untranslated region (3′-UTR) of target messenger RNAs. MiR-214-5p is a newly identified miRNA with its biological role largely unknown. In this study, we explored miR-214-5p expression status in 78 paired tumor and nontumor tissues obtained from patients with hepatocellular carcinoma (HCC) by RT-qPCR. The effects of miR-214-5p expression on HCC cell proliferation, cell cycle progression, and cell migration were measured by CCK-8 assay, flow cytometry, and wound-healing assay. A dual-luciferase activity assay was performed to identify whether KLF5 was a target of miR-214-5p. Kaplan-Meier curve and log-rank test were used to investigate the effects of miR-214-5p and KLF5 on overall survival and disease-free survival of patients with HCC. We found miR-214-5p expression was sharply reduced in HCC tissues and cell lines compared with the normal tissues and cell lines. Functional assay revealed that miR-214-5p overexpression could downregulate cell proliferation, cell migration, and arrested cell cycle at G0/G1 phase. Further, we validated Krüppel-like factor 5 (KLF5) as a direct target of miR-214-5p, and was upregulated in HCC and inversely correlated with the expression of miR-214-5p. Moreover, we found the low expression of miR-214-5p and high expression of KLF5 were correlated with tumor size, tumor stage, and poorer 5-year overall survival and disease-free survival of patients with HCC. In conclusion, our results suggested miR-214-5p functions as a tumor suppressor through targeting KLF5 in HCC. Also, miR-214-5p and KLF5 were identified as potential prognostic markers and might be therapeutic targets in HCC.     

LncRNA GAS5 regulates vascular smooth muscle cell cycle arrest and apoptosis via p53 pathway

Vascular remodeling is a pathological process following cardiovascular intervention. Vascular smooth muscle cells (VSMC) play a critical role in the vascular remodeling. Long noncoding RNAs (lncRNA) are a class of gene regulators functioning through various mechanisms in physiological and pathological conditions. By using cultured VSMC and rat carotid artery balloon injury model, we found that lncRNA growth arrest specific 5 (GAS5) serves as a negative regulator for VSMC survival in vascular remodeling. By manipulating GAS5 expression via adenoviral overexpression or short hairpin RNA knockdown, we found that GAS5 suppresses VSMC proliferation while promoting cell cycle arrest and inducing cell apoptosis. Mechanistically, GAS5 directly binds to p53 and p300, stabilizes p53-p300 interaction, and thus regulates VSMC cell survival via induction of p53-downstream target genes. Importantly, local delivery of GAS5 via adenoviral vector suppresses balloon injury-induced neointima formation along with an increased expression of p53 and apoptosis in neointimal SMCs. Our study demonstrated for the first time that GAS5 negatively impacts VSMC survival via activation the p53 pathway during vascular remodeling.     

Regulation of Expression of Deoxyhypusine Hydroxylase (DOHH), the Enzyme That Catalyzes the Activation of eIF5A,by miR-331-3p and miR-642-5p in Prostate Cancer Cells

The enzyme deoxyhypusine hydroxylase (DOHH) catalyzes the activation of eukaryotic translation initiation factor (eIF5A), a protein essential for cell growth. Using bioinformatic predictions and reporter gene assays, we have identified a 182-nt element within the DOHH 3′-untranslated region (3′-UTR) that contains a number of target sites for miR-331-3p and miR-642-5p. Quantitative RT-PCR studies demonstrated overexpression of DOHH mRNA and underexpression of miR-331-3p and miR-642-5p in several prostate cancer cell lines compared with normal prostate epithelial cells. Transient overexpression of miR-331-3p and/or miR-642-5p in DU145 prostate cancer cells reduced DOHH mRNA and protein expression and inhibited cell proliferation. We observed synergistic growth inhibition with the combination of miR-331-3p and miR-642-5p and mimosine, a pharmacological DOHH inhibitor. Finally, we identified a significant inverse relationship between the expression of miR-331-3p or miR-642-5p and DOHH in a cohort of human prostate cancer tissues. Our results suggest a novel role for miR-331-3p and miR-642-5p in the control of prostate cancer cell growth via the regulation of DOHH expression and eIF5A activity.