MicroRNA-222调控HUVEC c-kit基因表达及对内皮微粒的影响

The influence of microRNA-222 on regulation of the expression of c-kit and endothelial microparticles in human umbilical vein endothelial cells

目的:探讨微小RNA(miR)-222及特异性miR-222抑制剂对人脐静脉内皮细胞(HUVEC)c-kit基因的mRNA和蛋白表达、内皮微粒的影响。方法:设计miR-222-3p mimic和miR-222-3p inhibitor及相应的阴性对照(mimic control和inhibitor control),分别转染HUVEC,通过实时荧光定量聚合酶链式反应技术(qRT-PCR)验证转染效率。利用qRT-PCR检测c-kit基因mRNA表达水平;免疫印迹技术分析c-kit蛋白表达水平;CD144标记内皮微粒,流式细胞仪检测miR-222转染对内皮微粒分泌的影响。结果:与mimic control转染组相比,转染miR-222-3pmimic均显著降低c-kit基因mRNA和蛋白表达水平。与inhibitor control转染组相比,转染miR-222-3p inhibitor均显著提高c-kit基因mRNA和蛋白表达水平。流式细胞仪检测显示miR-222-3p表达上调导致内皮微粒分泌增加,miR-222-3p表达下调导致内皮微粒分泌减少。结论:miR-222表达上调抑制c-kit基因mRNA的表达,进而抑制蛋白的表达,miR-222表达上调可能通过引起HUVEC株细胞活化而致内皮微粒分泌增加,进而介导了内皮细胞功能的紊乱。

Objective: To explore the effect of miR-222 and specific miR-222 inhibitor on the expression of mRNA and protein of c-kit gene and its effect on endothelial microparticles in human umbilical vein endothelial cell(HUVEC) line. Methods: MiR-222-3p mimic,mimic negative control, miR-222-3p inhibitor and inhibitor negative control were synthesized and transfected into HUVEC line via transfection reagent Lipofectamine TM 2000 and the expressions of miR-222 in HUVEC were detected by real time quantitative PCR (qRT-PCR). The expression of mRNA and protein of c-kit gene were detected by qRT-PCR and Western blotting(WB). The influence on endothelial microparticles was detected by flow cytometry. Results: The overexpression of miRNA-222 in HUVEC line strongly downregulated the expression of mRNA and protein of c-kit gene in contrast with the mimic negative control group, in addition induced the secretion of endothelial microparticles. On the contrary, the expression of miR-222 signicantly enhanced the expression of mRNA and protein of c-kit gene in contrast with the inhibitor negative control group, in addition increased the secretion of endothelial microparticles. Conclusion: MiR-222 and c-kit gene and protein expression was negatively correlated. MiR-222 up-regulation may induce the activation of HUVEC, and increase the secretion of endothelial microparticles, which may leads to the dysfunction of endothelial cells.