黄芩苷通过CDH5/miR-125b-5p信号通路抑制Hcy诱导的VSMCs增殖和迁移机制研究

Study on mechanism of baicalin inhibits proliferation and migration of the VSMCs via regulating the CDH5/miR-125b-5p pathway

目的 探讨黄芩苷抑制同型半胱氨酸(Hcy)诱导的血管内皮细胞增殖和迁移机制。方法 采用TargetScan、miRTarBase软件预测miR-125b-5p与血管内皮钙粘蛋白(CDH5)的靶向关系;MTT法评价黄芩苷对Hcy诱导的血管平滑肌细胞(VSMCs)增殖的抑制作用;细胞划痕(Transwell)实验检测VSMCs的迁移能力;蛋白印迹(Western Blot)检测黄芩苷对CDH5、抗增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)蛋白表达的影响;实时荧光定量聚合酶链式反应(PCR)技术检测miR-125b-5p的表达;双荧光素酶报告基因检测技术验证miR-125b-5p与CDH5的靶向关系。结果 黄芩苷呈浓度依赖性抑制Hcy诱导的VSMCs增殖和迁移,并降低细胞增殖相关蛋白CDH5、PCNA、Cyclin D1的表达;黄芩苷可诱导miR-125b-5p基因的表达。双荧光素酶实验结果显示, miR-125b-5p类似物可显著抑制野生型CDH5-UTRwt的荧光酶活性,而对突变型CDH5-UTRmut无活性。结论 黄芩苷通过CDH5/miR-125b-5p信号通路抑制Hcy诱导的VSMCs增殖和迁移。

Objective To discuss the mechanism of baicalin inhibiting proliferation and migration of vascular endothelial cells induced by homocysteine(Hcy). Methods TargetScan and miRTarBase software were used to predict the targeting relationship between miR-125b-5p and vascular endothelial cadherin(CDH5);MTT assay was used to evaluate the inhibitory effect of baicalin on the proliferation of vascular smooth muscle cells(VSMCs) induced by Hcy;cell scratch(Transwell) test was used to detect the migration ability of VSMCs;Western blot was used to detect the effect of baicalin on the expression of CDH5, anti proliferating cell nuclear antigen(PCNA) and cyclin D1;the real-time fluorescence quantitative polymerase chain reaction(PCR) was used to detect the expression of miR-125b-5p;the targeting relationship between miR-125b-5p and CDH5 was verified by dual luciferase reporter gene detection technology. Results Baicalin inhibited the proliferation and migration of the Hcy-induced VSMCs with a concentration-dependent manner and down-regulated the proliferation-related proteins level of CDH5, PCNA, Cyclin D1;Baicalein induced the expression of miR-125b-5p gene;the results of dual luciferase assay showed that miR-125b-5p analogue significantly inhibited the luciferase activity of wildtype CDH5-UTRwt, while it was inactive against mutant CDH5-UTRmut. Conclusion Baicalein inhibits Hcyinduced proliferation and migration of VSMCs via the CDH5/miR-125b-5p signaling pathway.