沉默EZH2表达抑制胆管癌QBC939细胞增殖及其机制

摘要

目的:探讨沉默EZH2基因表达对胆管癌QBC939细胞增殖的抑制作用及其机制.方法:设计并合成靶向EZH2的siRNA体外转染QBC939细胞,以实时荧光定量PCR和Western Blot法检测mRNA及蛋白表达水平,通过MTT法和平板克隆实验检测细胞增殖能力,流式细胞术检测细胞凋亡及周期分布,β-半乳糖苷酶染色法检测细胞衰老情况,以Western Blot法检测H3K27me3、P14ARF、P16INK4a、P53、P21、E2F1蛋白表达变化.结果:靶向siRNA可有效沉默EZH2表达,显著抑制细胞体外增殖能力.与对照组相比,实验组细胞G1/S期比值显著升高,出现G1期阻滞,而凋亡率未见明显差异;β-半乳糖苷酶染色阳性,呈衰老特征;衰老通路的P14ARF、P16INK4a、P53、P21蛋白表达上调,而H3K27me3、E2F1蛋白表达下调.结论:沉默EZH2表达可显著抑制QBC939细胞的体外增殖能力,其机制可能与调控衰老通路有关.%Objective To investigate the inhibitory effects of silencing expression ofEZH2 gene on the cell proliferation of human QBC939 cells and its mechanisms. Methods The targeting siRNA was designed and trans-fected into QBC939cells. The expressions of EZH2 mRNA and protein were detected by real-time qPCR and west-ern blotting,respectively. The ability of cellproliferationwas analyzed by MTT assay and plate clone formation assay. Cell apoptosis and cycle percentage weremeasured by flow cytometry. Cell senescence was assessed byβ-galactosi-dase dyeing.The expressions of H3K27me3,P14ARF,P16INK4a,P53,P21 and E2F1 proteinwere determined by West-ern blotting.Results Compared with the control group ,the expressions of mRNAand protein were significantly elevat-ed in experimental group. The ability of cellproliferation in the experiment group was significantly down regulated , which could also cause a rise of G1/S phase ,but not a marked variation of apoptosis rate. Silencing EZH2 would induce a obvious senescence phenotype in QBC939 cells. EZH2-siRNA transferredcould also down-regulate the expressions of H3K27me3 and E2F1 protein,while up-regulating the expressions of P14ARF,P16INK4a,P53 and P21 protein in QBC939 cells.Conclusions Silencing EZH2 could induce a significant inhibition on cell proliferation of QBC939 cells,the mechanism of which may be associated with the senescence pathway regulation.