miR-148-3p抑制剂下调Akt2调控HUVECs细胞增殖及凋亡机制研究

Mechanism of miR-148-3p inhibitor in down-regulating Akt2 and regulating HUVECs cell proliferation and apoptosis

目的探究微RNA(miR)-148-3p对模拟角膜新生血管形成常见体外细胞模型人脐静脉内皮细胞(HUVECs)的增殖及凋亡的影响。方法实验分2个阶段,第1阶段,HUVECs分为:空白对照组(以HUVECs不作任何处理)、NC inhibitor组(转染NC inhibitor)、miR-148-3p inhibitor组(转染miR-148-3p inhibitor)。采用CCK-8法检测抑制miR-148-3p表达对HUVECs增殖能力的影响;TargetScan筛选miR-148-3p潜在靶基因,双荧光素酶报告基因实验进行验证;蛋白免疫印迹法(Western blot)检测Akt2表达水平变化;第2阶段,HUVECs分为:NC组(转染过表达质粒Akt2的阴性对照)、Akt2组(转染过表达Akt2质粒);NC inhibitor组、miR-148-3p inhibitor组、miR-148-3p inhibitor+Akt2组(共转染miR-148-3p inhibitor和过表达Akt2质粒),采用CCK-8法检测miR-148-3p inhibitor和Akt2对HUVECs增殖能力的影响;采用末端DNA转移酶dUPT缺口末端标记(TUNEL)法检测各组细胞凋亡情况。结果常规培养48、72、96 h,miR-148-3p inhibitor组中HUVECs增殖能力低于NC inhibitor组和空白对照组,差异有统计学意义(P<0.05);miR-148-3p inhibitor组pMIR-Akt2-wt及pMIR-Akt2-Mut质粒荧光素酶活性与NC inhibitor组比较,差异无统计学意义(P>0.05),miR-148-3p inhibitor组Akt2表达水平低于NC inhibitor组(P<0.05);与NC组比较,Akt2组细胞增殖能力升高,凋亡指数(AI)值降低;常规培养48、72、96 h,与NC inhibitor组比较,miR-148-3p inhibitor组增殖能力降低,AI值升高;与miR-148-3p inhibitor组比较,miR-148-3p inhibitor+Akt2组增殖能力升高,AI值降低,差异均有统计学意义(P<0.05)。结论miR-148-3p抑制剂可能通过下调Akt2抑制HUVECs增殖并促进凋亡。

Objective To investigate the effect of miR-148-3p on the proliferation and apoptosis of human umbilical vein endothelial cells(HUVECs)which was the common in vitro cell model simulating corneal neovascularization.Methods The experiment was divided into the two stages,in the first stage:HUVECs cells were divided into the blank control group(HUVECs cells without any modification),the NC inhibitor group(transfected with NC inhibitor),and the miR-148-3p inhibitor group(transfected with miR-148-3p inhibitor).The effect of inhibiting miR-148-3p expression on the proliferation of HUVECs was detected by CCK-8 assay.TargetScan was used to screen the miR-148-3p potential target genes.Double luciferase reporter assay used to validate.Western blot was used to detect the change of expression level of Akt2 protein.In the second stage:HUVECs were divided into the NC group(transfected with negative control of over-expression plasmid of Akt2),the Akt2 group(transfected with overexpression plasmid of Akt2),the NC inhibitor group,miR-148-3p inhibitor group,miR-148-3p inhibitor+Akt2 group(co-transfected with miR-148-3p inhibitor and overexpression Akt2 plasmid of Akt2).The effect of miR-148-3p inhibitor and Akt2 on the HUVECs prolif-eration ability were detected by the CCK-8 assay.The apoptosis ability of each group was detected by TUNEL assay.Results After 48,72,96 h of common culture,The HUVECs cell proliferation ability in the miR-148-3p inhibitor group was lower than that in the NC inhibitor group and blank control group,and the difference were statistically significant(P<0.05).There was no statistically significant difference in the luciferase activity of pMIR-Akt2-WT plasmid between the miR-148-3p inhibitor group and the NC inhibitor group(P>0.05).The protein expression level of Akt2 in the miR-148-3p inhibitor group was lower than that of the NC inhibitor group(P<0.01).Compared with the NC group,the cellular proliferation ability in the Akt2 group was increased and the AI value was decreased.Compared with the NC inhibitor group,the proliferation ability in the miR-148-3p inhibitor group was decreased and the apoptosis index(AI)value was increased.Compared with the miR-148-3p inhibitor group,the proliferation ability in the miR-148-3p inhibitor+Akt2 group was increased and the AI value was decreased,and the differences were statistically significant(P<0.05).Conclusion The miR-148-3p inhibitor may inhibit the proliferation and promote apoptosis in HUVECs cell lines by regulating Akt2.