miR-199b促进慢性创面血管化的作用及机制研究

Study on the Role and Mechanism of miR-199b in Promoting Vascularization of Chronic Wound

目的:探索miR-199b 对HaCaT细胞增殖、迁移及对VEGFA、JAG1、SET信号通路的影响。方法:转染miR-199b 上调与下调慢病毒到人永生化表皮细胞(HaCaT)。分为miR-199b 上调(up)、miR-199b 下调(dowm)、空载对照(NC组)三个组,进行CCK8、凋亡、周期试验,并采用RT-PCR技术检测VEGFA、JAG1、SET基因的表达,采用WB技术检测miR-199b 对下游蛋白变化(如VEGFA、JAG1等分子)的影响。结果:miR-199b up组OD450/fold值显著高于NC组,差异有统计学意义(P<0.05)、miR-199b down组OD450/fold值略低于NC组,差异无统计学意义(P>0.05)。与NC组相比,G1期miR-199b up组与miR-199b down组的细胞增多(P<0.05);在S期,miR-199b up组及miR-199b down组的细胞减少(P<0.05);在G2/M期,miR-199b up组及miR-199b down组的细胞减少,差异均具有统计学意义(P<0.05)。与NC组比较,miR-199b up组凋亡细胞百分比下降(P<0.05)、miR-199b down组凋亡细胞百分比上升(P<0.05);SET在miR-199b up组的表达明显低于NC组,在miR-199b down组表达明显高于NC组。从定量PCR结果可以看出,HaCaT细胞中,miR-199b up组JAG1表达丰度略高于NC组,差异无统计学意义(P>0.05),miR-199b down组JAG1表达丰度明显高于NC组(P<0.05);miR-199b up组VEGFA表达丰度明显低于NC组(P<0.05)、miR-199b down组VEGFA表达丰度明显高于NC组(P<0.05);miR-199b up组SET表达丰度小于NC组(P>0.05)、miR-199b down组SET表达丰度明显大于NC组(P<0.05)。结论:miR-199b 可抑制VEGF、JAG1、SET蛋白的表达,为其靶基因,可促进细胞的增殖,延长细胞周期,并抑制细胞凋亡。

Objective To explore the effects of miR-199b on proliferation and migration of HaCaT cells,as well as VEGFA,JAG1 and SET signaling pathways.Methods Transfection of miR-199b mimics and inhibitor lentivirus into human immortalized epidermal cells HaCaT was performed.The study was divided into three groups:miR-199b up,miR-199b down and no-load control.CCK8,cycle and scratch test were conducted,and the expression of VEGFA,JAG1 and SET genes were detected by RT-PCR,and the downstream protein changes(such as VEGFA,JAG1 and other molecules)were detected by WB technology.Results The OD450/fold value of miR-199b up group was significantly higher than that of NC group(P<0.05).The OD450/fold value of miR-199b down group was slightly lower than that of NC group(P>0.05).In the G1 phase,cells in the miR-199b up and down group were increased compared to the control group(P<0.05).At the S stage,compared with the control group,the cells in the miR-199b up and down group were reduced(P>0.05).In the G2/M phase,compared with the control group,the cells in the miR-199b up and down group were reduced(P>0.05).Compared with the NC group,the percentage of apoptotic cells in the miR-199b up group decreased(P<0.05)and the percentage of apoptotic cells in the miR-199b down group increased(P<0.05).The expression of SET was significantly lower in the miR-199b up group than in the NC group,and significantly higher in the miR-199b down group than in the NC group.According to the quantitative PCR results,the expression abundance of JAG1 in the miR-199b up group was slightly higher than that in the NC group(P>0.05).The expression abundance of JAG1 in the miR-199b down group was significantly higher than that in the NC group(P<0.05).The expression abundance of VEGFA in the miR-199b up group was significantly lower than that in the NC group(P<0.05).The expression abundance of VEGFA in the miR-199b down group was significantly higher than that in the NC group(P<0.05).SET expression abundance of miR-199b up group was smaller than that of NC group(P>0.05).The expression abundance of SET in the miR-199b down group was significantly greater than that in the NC group(P<0.05).Conclusion miR-199b is the target gene of VEGF,JAG1 and SET proteins by inhibiting their expression.It can promote cell proliferation,prolong cell cycle and inhibit cell apoptosis.