论文部分内容阅读
目的探讨miRNA-143(miR-143)和DNMT3a在血管平滑肌细胞增殖中的作用及机制。方法分别用miR-143的前体和抑制物干预血管平滑肌细胞(VSMCs),MTT法检测VSMCs增殖情况,qRT-PCR法检测miR-143和DNMT3a的表达,Western blot法检测DNMT3a的蛋白表达,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,双荧光素酶报告基因检测系统分析荧光素酶活性。结果 miR-143的前体干预VSMCs后,细胞的增殖活性显著下降,DNMT3a mRNA和蛋白表达均降低,而miR-143的抑制物作用后细胞增殖活性明显增强,DNMT3a表达增加;转染miR-143前体后,荧光素酶活性减弱,而用miR-143抑制物作用细胞后,荧光素酶活性明显增强(P<0.01);干扰DNMT3a后,miR-143的表达增加,但miR-143启动子区甲基化程度降低。结论 miR-143和DNMT3a相互调控可能是VSMCs增殖的重要机制。
Objective To investigate the role of miRNA-143 (miR-143) and DNMT3a in the proliferation of vascular smooth muscle cells. Methods The proliferation and proliferation of VSMCs were detected by MTT assay. The expression of miR-143 and DNMT3a was detected by qRT-PCR. The expression of DNMT3a protein was detected by Western blot. The methylation status of the miR-143 promoter region was analyzed by the type-specific down-stream methylation PCR and the luciferase activity was analyzed by the dual luciferase reporter assay system. Results The proliferation of VSMCs was significantly decreased and the expression of both DNMT3a mRNA and protein was decreased after miR-143 pretreatment. However, miR-143 inhibited the proliferation of VSMCs and increased the expression of DNMT3a. The luciferase activity was decreased after treated with miR-143 inhibitor, while the luciferase activity was significantly increased after cells were treated with miR-143 inhibitor (P <0.01). The expression of miR-143 was increased after interference with DNMT3a, but miR-143 promoter District methylation decreased. Conclusion The mutual regulation of miR-143 and DNMT3a may be an important mechanism of VSMCs proliferation.