miR-206对肺动脉内皮细胞增殖的影响及机制研究

The role of microRNA-206 in the proliferation of human Pulmonary artery endothelial cells and its primary mechanism

目的:探讨microRNA-206(miR-206)对人肺动脉内皮细胞(human pulmonary artery endothelial cells,HPAECs)增殖能力的影响及其作用机制。方法:体外培养HPAECs细胞株后,分别进行miR-206 agomir/antagomir转染,采用实时荧光定量聚合酶链反应(qRT-PCR)验证转染效果。对转染成功后的HPAECs采用CCK-8法、EdU法检测细胞活性和增殖能力,蛋白质印迹法(Western Blot)检测JAK2-STAT3信号通路蛋白质表达。以JAK2抑制剂AG490(100 nM)处理HPAECs,采用CCK-8法检测细胞增殖能力的变化,酶联免疫吸附实验(ELISA)检测HPAECs中血管内皮生长因子(vascular endothelial growth factor,VEGF)表达水平的变化。结果:miR-206 antagomir组HPAECs在转染48 h、72 h时细胞增殖能力高于Con组、agomir-NC组及antagomir-NC组,差异均具有统计学意义(P<0.05)。HPAECs在miR-206 antagomir转染后48 h增殖率、磷酸化STAT3、磷酸化JAK2表达量高于Con组和antagomir-NC组,差异均具有统计学意义(P<0.05)。miR-206 antagomir组VEGF含量显著上升,高于Con组、antagomir-NC和AG 490+miR-206 antagomir组,差异均具有统计学意义(P<0.05)。AG 490处理后miR-206 antagomir组HPAECs细胞增殖能力与Con组及antagomir-NC组比较,差异均无统计学意义(P>0.05)。结论:(1)miR-206 antagomir促进HPAECs增殖。(2)抑制HPAECs miR-206转录可以上调磷酸化JAK2及STAT3蛋白表达,该过程能被JAK2-STAT3信号通路抑制剂部分逆转。(3)miR-206 antagomir对HPAECs增殖能力的影响可能与促进VEGF分泌相关。

Objective:To discuss and probe into the effect of microRNA-206(miR-206)on the proliferation of human pulmonary artery endothelial cells(HPAECs)and its primary mechanism.Methods:After cultured in vitro,HPAECs were transfected with miR-206 agomir/antigomir,and the transfection efficiency was verified by real-time PCR.The cell counting kit-8(CCK-8)and Edu(5-ethylyl-2′-deoxyuridine)methods were used to detect the cell activity and proliferation of HPAECs.The protein expression of JAK2-STAT3 signaling pathway was detected by Western blot(WB).The HPAECs were treated with JAK2 inhibitor AG490(100 nm).The proliferation of HPAECs was detected by CCK-8.The secretion of vascular endothelial growth factor(VEGF)in HPAECs was detected by enzyme-linked immunosorbent assay(ELISA).Results:After HPAECs were successfully transfected,the proliferation ability of HPAECs in miR-206 antagomir group was significantly higher than that of the other experimental groups at 48 hours and 72 hours(P<0.05).After miR-206 antagomir transfection for 48 h,the proliferation rate of HPAECs increased significantly(P<0.05),the expression of phospho STAT3 and phospho JAK2 increased significantly(P<0.05).Compared with the control group,there was no significant difference in the activity of HPAECs incubated with AG490(100 nm)2 hours in advance after transfection with miR-206 antagomir(P>0.05),and there was no significant difference in the expression of VEGF(P>0.05).Conclusions:(1)miR-206 antagomir could promote the proliferation of HAPECs.(2)Inhibition of miR-206 in HPAECs could regulate the expression of JAK2-STAT3 signal pathway phosphorylated JAK2 and STAT3 protein.The proliferation promoting effect of miR-206 antagomir on HPAECs could be reversed by the inhibitor of JAK2-STAT3 signal pathway.(3)The effect of miR-206 antagomir on the proliferation of HPAECs might be related to the enhancement of VEGF secretion.