miR-130a对大鼠脑基底动脉平滑肌细胞活力和凋亡的影响

目的:探讨微小RNA(miR)-130a对大鼠脑基底动脉平滑肌细胞(basilar arterial smooth muscle cells,BASMCs)生物学行为的影响及可能的作用机制。方法:采用real-time PCR法检测大鼠BASMCs在血管紧张素Ⅱ(AngⅡ)作用下miR-130a的表达水平。转染miR-130a inhibitor下调BASMCs中miR-130a的表达,采用CCK-8法和流式细胞术检测BASMCs活力、细胞周期及凋亡情况;Western blot检测细胞周期及凋亡相关调控因子细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖性激酶2(CDK2)、p21、p-Rb、Bcl-2和cleaved caspase-3/caspase-3的蛋白水平。Real-time PCR及Western blot检测检测生长阻滞特异性同源盒蛋白(Gax)的表达情况。结果:AngⅡ可促进BASMCs中miR-130a的表达,而抑制Gax的表达。miR-130a inhibitor可部分抑制AngⅡ增加BASMCs细胞活力的效应,并上调Gax的表达。此外,下调miR-130a后细胞早期凋亡率显著增加(P0.05);同时细胞中cyclin D1、CDK2、Bcl-2和p-Rb的蛋白水平均显著降低,p21及cleaved caspase-3的蛋白水平显著升高(P0.05)。结论:沉默miR-130a可上调BASMCs中Gax的表达,进而影响细胞周期及凋亡相关因子的表达,从而抑制细胞活力,并促进其凋亡,提示miR-130a可作为高血压脑血管重构的一个潜在诊疗靶点。

Effects of miR-130a on viability and apoptosis of rat basilar arterial smooth muscle cells

AIM: To investigate the regulatory effects of microRNA (miR)-130a on the biological characteristics of rat basilar arterial smooth muscle cells (BASMCs) and its underlying mechanisms. METHODS: The expression of miR-130a in rat BASMCs was measured by real-time PCR. After knockdown of miR-130a with inhibitor in the BASMCs, the cell viability, cell cycle distribution and apoptosis were detected by CCK-8 assay and flow cytometry. The expression of cell cycle-and apoptosis-related molecules, such as cyclin D1, cyclin-dependent kinase 2 (CDK2), p21, Bcl-2 and cleaved caspase-3/caspase-3 at protein levels was determined by Western blot. The growth arrest-specific homeobox protein (Gax) expression at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: AngiotensionⅡ (AngⅡ) up-regulated the expression of miR-130a and down-regulated the expression of Gax (P<0.05). Transfection with miR-130a inhibitor partly reversed the increase in AngⅡ-induced cell viability and promoted the Gax expression. Furthermore, the early cell apoptotic rate was significantly increased after down-regulation of miR-130a (P<0.05), and the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb were significantly decreased, accompanied with the up-regulation of p21 and cleaved caspase-3 (P<0.05). CONCLUSION: Down-regulation of miR-130a restrains the viability and promotes the apoptosis of BASMCs by promoting Gax expression and regulating cell cycle-and apoptosis-related molecules, suggesting that miR-130a may be a potential therapeutic target of brain vascular remodeling during hypertension.