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目的探讨miRNA-143在同型半胱氨酸(homocysteine,Hcy)致血管平滑肌细胞增殖中的作用。方法原代培养血管平滑肌细胞(vascular smooth muscle cells,VSMCs),用不同浓度的Hcy和叶酸干预,MTT法检测VSMCs增殖情况,qRT-PCR法检测miR-143的表达,巢式降落式特异性甲基化PCR法分析miR-143启动子区的甲基化状态,分别用miR-143的前体和抑制物转染细胞检测细胞增殖。结果不同浓度的Hcy均可以引起VSMCs增殖活性增强,miR-143的mRNA表达降低,miR-143启动子区甲基化程度升高,以100μmol·L~(-1)Hcy最为明显(P<0.01),miR-143前体作用VSMCs后,细胞的增殖活性减弱(P<0.01),细胞转染miR-143的抑制物后细胞增殖活性明显增强(P<0.01)。结论 miR-143可以抑制VSMCs增殖,其机制可能与miR-143启动子区高甲基化有关。
Objective To investigate the role of miRNA-143 in the proliferation of vascular smooth muscle cells induced by homocysteine (Hcy). Methods Primary cultured vascular smooth muscle cells (VSMCs) were treated with different concentrations of Hcy and folic acid. The proliferation of VSMCs was detected by MTT assay. The expression of miR-143 was detected by qRT-PCR, The methylation status of miR-143 promoter region was analyzed by PCR. The cell proliferation was detected by transfecting cells with miR-143 precursor and inhibitor respectively. Results Hcy at different concentrations increased the proliferative activity of VSMCs, decreased the mRNA expression of miR-143, and increased the methylation level of miR-143 promoter, with the most significant difference being Hcy at 100 μmol·L -1 (P <0.01) ), the proliferative activity of VSMCs decreased after miR-143 pre-treated with VSMCs (P <0.01). The proliferation of VSMCs transfected with miR-143 was significantly enhanced (P <0.01). Conclusion miR-143 can inhibit the proliferation of VSMCs, which may be related to the hypermethylation of miR-143 promoter region.