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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 micro RNA-34a (mi R-34a) in the proliferation of vascular smooth muscle cells (VSMCs) induced by low shear stress. Methods Normal parallel stress (NSS) and low shear stress (Low SSS) of 1.5 Pa were applied to endothelial cells (ECs) co-cultured with VSMCs by using cell platelet culture system. ), Loading time is 12 h. The proliferating cell nuclear antigen (PCNA) protein expression of VSMCs was detected by Western blotting to reflect the proliferation of VSMCs. Real-time PCR was used to detect the expression of mi R-34a in co-cultured VSMCs. The downstream targets of mi R-34a were predicted by Target Scan and mi RWalk. Western blotting was used to detect the expression of Forkhead transcription factor J2 (Foxj2), a mi R-34a target of co-cultured VSMCs. The expression of mi R-34a in VSMCs was up-regulated and downregulated by mimics and inhibitor respectively. The expression of Foxj2 and PCNA was detected by Western blotting to verify the regulation of mi R-34a and Foxj2. Results Low SS promoted PCNA expression in VSMCs co-cultured with NSS and significantly up-regulated mi R-34a expression in co-cultured VSMCs. The downstream target protein of mi R-34a is Foxj2 predicted by Target Scan and mi RWalk. The expression of Foxj2, a mi R-34a target protein, was significantly reduced in VSMCs incubated with Low SS. Under static condition, the expression of mi R-34a was up-regulated in VSMCs, and the expression of Foxj2 was significantly decreased and the expression of PCNA was significantly increased. The expression of mi R-34a and the expression of Foxj2 in VSMCs were significantly decreased and the expression of PCNA was significantly decreased. Conclusion Low SS can promote the proliferation of VSMCs through regulating the mi R-34a and the target protein Foxj2 in VSMCs. The results provide new mechanistic biology experimental basis for further elucidating the pathogenesis of atherosclerotic disease and drug treatment targets.