microRNA-34a在低切应力诱导血管平滑肌细胞增殖中的作用

The role of microRNA-34a in low shear stress-induced proliferation of vascular smooth muscle cells

目的探讨micro RNA-34a(mi R-34a)在低切应力诱导血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖中的作用。方法应用细胞联合培养平行平板流动腔系统对与VSMCs联合培养的内皮细胞(endothelial cells,ECs)施加1.5 Pa正常切应力(normal shear stress,NSS)和0.5 Pa低切应力(low shear stress,Low SS),加载时间为12 h。Western blotting检测联合培养VSMCs的增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白表达,以此反映VSMCs的增殖能力。实时PCR检测联合培养VSMCs的mi R-34a表达变化。通过Target Scan、mi RWalk等网站预测mi R-34a的下游靶蛋白。Western blotting检测联合培养VSMCs的mi R-34a靶蛋白Forkhead转录因子J2(forkhead box j2,Foxj2)表达。通过mimics和inhibitor转染技术,分别上调和抑制VSMCs的mi R-34a表达,Western blotting检测Foxj2及PCNA的表达变化,验证mi R-34a和Foxj2之间的调控关系。结果与NSS相比,Low SS促进联合培养VSMCs的PCNA表达,并显著上调联合培养VSMCs的mi R-34a表达。通过Target Scan、mi RWalk等网站预测mi R-34a的下游靶蛋白为Foxj2。Low SS加载下联合培养VSMCs的mi R-34a靶蛋白Foxj2表达明显降低。静态条件下上调VSMCs的mi R-34a表达,靶蛋白Foxj2表达明显降低,PCNA表达显著升高;抑制VSMCs的mi R-34a表达,靶蛋白Foxj2表达明显上调,PCNA表达显著降低。结论 Low SS通过调控联合培养VSMCs的mi R-34a和靶蛋白Foxj2,促进VSMCs增殖。研究结果为进一步阐明动脉粥样硬化疾病的发病机制及药物治疗靶标提供新的力学生物学实验依据。

Objective To investigate the role of microRNA-34a （miR-34a） in the proliferation of vascular smooth muscle cells （VSMCs） induced by low shear stress （LowSS）. Methods Using co-culture parallel plate flow chamber system, endothelial cells （ECs） and VSMCs were co-cultured and applied with normal shear stress （1.5 Pa） and LowSS （0.5 Pa） for 12 h. The expression of proliferating cell nuclear antigen （PCNA） in the co- cultured VSMCs was detected by Western blotting to determine the proliferation capacity of VSMCs. Real-time PCR was used to examine the miR levels of miR-34a in the co-cultured VSMCs. The target proteins of miR-34a were predicted by TargetScan, miRWalk and some other websites. Western blotting was used to detect expres- sion of Forkhead box j2 （Foxj2） in the co-cultured VSMCs. Mimics and inhibitor were used to up-regulate or in-hibit the expression of miR-34a, and then the expression of Foxj2 and PCNA was detected by Western blotting to verify the regulation relationship between miR-34a and Foxj2. Results Compared with NSS, LowSS promoted the PCNA expression and significantly up-regulated the miR-34a expression in the co-cultured VSMCs, Foxj2 was predicted to be the downstream target protein of miR-34a by TargetScan, miRWalk and some other websites. Foxj2 expression decreased significantly in the co-cultured VSMCs under LowSS application. Under static condi- tion, the expression of Foxj2 obviously decreased and the expression of PCNA obviously increased by up-regula- ting miR-34a expression in VSMCs. While inhibiting the expression of miR-34a in VSMCs would result in a signifi- cant increase in the expression of Foxj2 and a significant decrease in the expression of PCNA. Cenclusiens LowSS can promote the proliferation of VSMCs by regulating miR-34a and target protein Foxj2 in the co-cultured VSMCs. This research finding will provide new mechanobiological experimental reference for further illustrating the pathogenesis of atherosclerosis and finding the therapeutic targets for drugs.proliferation