miR-let-7b-5p靶向调控LIMD2对胃癌细胞间质-上皮转化的影响

Effect of miR-let-7b-5p targeting LIMD2 on mesenchymal-epithelial transition of gastric cancer cells

目的:探究微小RNA(miR)-let-7b-5p靶向调控含LIM域2蛋白(LIMD2)对胃癌(GC)细胞间质-上皮转化(MET)的影响。方法:采用qRT-PCR检测GES-1、SCG-7901、MGC-803、BGC-823细胞中miR-let-7b-5p相对表达水平;构建绿色荧光蛋白(GFP)和miR-let-7b-5p稳定过表达细胞株,qRT-PCR法检测miR-let-7b-5p过表达效率;生物信息学和双荧光素酶实验预测并验证miR-let-7b-5p与LIMD2靶向关系;构建pcDNA5-LIMD2过表达载体,将细胞分为NC组、miR-let-7b-5p组、miR-let-7b-5p-pcDNA5组和miR-let-7b-5p-LIMD2组,Western Blot检测各组细胞中LIMD2、Vimentin、N-cadherin、E-cadherin蛋白表达;Transwell法检测miR-let-7b-5p对MGC-803细胞侵袭能力的影响。结果:与GES-1相比,SCG-7901、MGC-803、BGC-823细胞中miR-let-7b-5p相对表达水平明显下降(P<0.05)。与NC组相比,miR-let-7b-5p组miR-let-7b-5p相对表达量升高(P<0.05),转染LIMD2-WT的细胞相对荧光素酶活性明显降低(P<0.05),LIMD2、Vimentin、N-cadherin蛋白表达下调(P<0.05),而E-cadherin蛋白表达上调(P<0.05),细胞侵袭个数减少(P<0.05)。与miR-let-7b-5p组相比,miR-let-7b-5p-LIMD2组LIMD2、Vimentin、N-cadherin蛋白表达上调(P<0.05),而E-cadherin蛋白表达下调(P<0.05),细胞侵袭个数增多(P<0.05)。结论:过表达miR-let-7b-5p可靶向下调LIMD2促进胃癌细胞的MET,从而有效改善肿瘤细胞的侵袭。

Objective:To explore the effect of microRNA(miR)-let-7b-5p targeting LIM domain containing 2(LIMD2)on mesenchymal-epithelial transition(MET)of gastric cancer(GC)cells.Methods:Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the relative expression level of miR-let-7b-5p in GES-1,SCG-7901,MGC-803,and BGC-823 cells;the green fluorescent protein(GFP)and miR-let-7b-5p stable overexpression cell lines were constructed,qRT-PCR method was used to detect the overexpression efficiency of miR-let-7b-5p;bioinformatics and dual-luciferase experiments were used to predict and verify the targeting relationship between miR-let-7b-5p and LIMD2;the pcDNA5-LIMD2 overexpression vector was constructed and the cells were divided into NC group,miR-let-7b-5p group,miR-let-7b-5p-pcDNA5 group and miR-let-7b-5p-LIMD2 group.Western Blot was used to detect the protein expression of LIMD2,Vimentin,N-cadherin and E-cadherin in each group of cells;and Transwell method was used to detect the influence of miR-let-7b-5p on the invasion ability of MGC-803 cells.Results:Compared with GES-1,the relative expression level of miR-let-7b-5p in SCG-7901,MGC-803,and BGC-823 cells was decreased(P<0.05).Compared with the NC group,the relative expression of miR-let-7b-5p in the miR-let-7b-5p group was increased(P<0.05),the relative luciferase activity of cells transfected with LIMD2-WT was reduced(P<0.05),the expression of LIMD2,Vimentin and N-cadherin proteins was down-regulated(P<0.05),the expression of E-cadherin protein was up-regulated(P<0.05),and the number of cell invasion was reduced(P<0.05).Compared with the miR-let-7b-5p group,the expression of LIMD2,Vimentin and N-cadherin proteins in the miR-let-7b-5p-LIMD2 group was up-regulated(P<0.05),the expression of E-cadherin protein was down-regulated(P<0.05),and the number of cell invasion was increased(P<0.05).Conclusion:Overexpression of miR-let-7b-5p can target down-regulation of LIMD2 to promote MET of gastric cancer cells,thereby effectively improving tumor cell invasion.