let-7g对子宫内膜癌RL-95-2细胞中RB1表达的调控

MicroRNA let-7g Regulates the Expression of RB1 in RL-95-2 Endometrial Carcinoma Cells

目的:探讨子宫内膜癌RL-95-2细胞中let-7g对其靶基因视网膜母细胞瘤相关蛋白1(retinoblastoma-associated protein 1,RB1)表达的调控作用。方法:运用生物信息学方法对let-7g进行靶基因预测并分析其靶基因RB1;let-7g模拟物(let-7g mimic)、模拟物对照组(mimic control)、let-7g抑制物(let-7g inhibitor)、抑制物对照组(inhibitor control)分别转染至RL-95-2细胞,采用CCK-8法检测其对细胞增殖的影响;实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)检测转染后RB1 mRNA的表达差异;Western blotting检测RB1蛋白的表达。结果:RB13'非编码区(3'UTR)含有一个let-7g结合位点,而且该结合位点在多个物种中高度保守;与未转染组相比,转染let-7g mimic可促进细胞增殖,且使RB1蛋白的表达显著降低,而转染let-7g抑制物则抑制细胞增殖,使RB1蛋白的表达显著增高,其余各组无明显变化;而各转染组RB1 mRNA表达无明显差异。结论:子宫内膜癌RL-95-2细胞中let-7g可以结合到RB1的3'UTR,负性调控RB1的表达,从而促进细胞生长。

Objective： To explore the regulatory effect of let-7g on expression of its target gene retinoblastomaassociated protein（RB1） in RL-95-2 endometrial carcinoma cells.Methods： Computational approaches were used for let-7g target prediction.Among all the predicted candidate targets,RB1 was chosen for further investigation.After let7g mimic,mimic control,inhibitor,inhibitor control were transfected into RL-95-2 cells,respectively;cell proliferation was determined by CCK-8 assay;the expressions of RB1 mRNA and protein were determined by RT-qPCR and Western blotting,respectively.Results： By bioinformatics analysis,RB1 3＇UTR contained a let-7g binding sites which is highly conserved in several species.Transfection with the let-7g mimic can promote cell proliferation,and decrease the RB1 protein level significantly,the opposite results were also observed in cells transfected with let-7g inhibitor,but not in cells transfected with mimics control or inhibitor control,when compared with RL-95-2 cells,however,there was no significant difference on RB1 mRNA level.Conclusion： let-7g can negatively regulate the expression of RB1 by binding to RB1 3＇UTR in RL-95-2 endometrial carcinoma cells,thus promoting cell growth.