长链非编码RNA CCAT2对结肠癌细胞增殖和凋亡的影响及机制探讨

The effects of lncRNA CCAT2 on the proliferation and apoptosis in colon carcinoma cell and its mechanism

目的:观察长链非编码RNA(lncRNA)结肠癌相关转录本2(CCAT2)在结肠癌细胞系中的表达及对细胞增殖和凋亡的影响,并探讨其机制。方法:采用荧光定量PCR技术(qRT-PCR)检测结肠癌细胞系HCT116、SW620、LOVO、HT29与正常结肠上皮细胞系NCM460中lncRNA CCAT2的表达。将结肠癌细胞系SW620分成CCAT2-siRNA组、Control-siRNA组及Mock组,CCAT2-siRNA组和Control-siRNA组经Lipofectamine^(TM) 2000分别转染CCAT2-siRNA及Control-siRNA,Mock组以PBST作对照。MTT实验和流式细胞术分别测定增殖和凋亡能力,Western blot测定p53、裂解型聚腺苷二磷酸-核糖聚合酶(PARP)及B细胞淋巴瘤/白血病-2(Bcl-2)蛋白的表达。结果:lncRNA CCAT2在结肠癌细胞系LOVO、HT29、HCT116、SW620中均比正常结肠上皮细胞系NCM460表达水平升高(P<0.05)。MTT示:转染后0 h、24 h、48 h,CCAT2-siRNA组与Control-siRNA组OD_(490 nm)值差异无统计学意义(P>0.05)。转染后72 h、96 h,CCAT2-siRNA组OD_(490 nm)值低于Control-siRNA组(P<0.05)。CCAT2-siRNA组细胞凋亡率高于Control-siRNA组(P<0.01)。与Control-siRNA组相比,CCAT2-siRNA组p53、裂解型PARP表达上调,Bcl-2蛋白表达下调。结论:lncRNA CCAT2在结肠癌细胞系中高表达,敲低lncRNA CCAT2表达可抑制结肠癌细胞增殖,并诱导凋亡,其机制可能与p53、裂解型PARP表达上调,Bcl-2蛋白表达下调有关。

Objective: To investigate the expression of lncRNA CCAT2 in colon carcinoma cell lines and its roles on proliferation and apoptosis of colon carcinoma in vitro,and explore the underlying mechanism. Methods: The expression of lncRNA CCAT2 in colon carcinoma cell lines including LOVO,HT29,HCT116,SW620 and normal colon cells was assayed by qRT-PCR. The SW620 cell line was divided into three groups and transfecte CCAT2-siRNA and Control-siRNA and PBST by LipofectamineTM2000,respectively. MTT assay and flow cytometry was used to measure the proliferation and apoptosis ability. The expression levels of p53,cleaved PARP and Bcl-2 protein was measured by Western blot. Results: Compared with NCM460 cell line,lncRNA CCAT2 was up-regulated expression in colon carcinoma cell lines,including LOVO,HT29,HCT116 and SW620( P < 0. 05). MTT showed that there was no significantly difference between CCAT2-siRNA and Control-siRNA group in terms of OD490 nmafter transfect for 0 h,24 h and 48 h( P > 0. 05). The OD490 nmvalue of CCAT2-siRNA group was significantly lower than that in Control-siRNA after transfect for 72 and 96 h( P < 0. 05). The apoptosis rate of CCAT2-siRNA group was significantly higher than that in Control-siRNA group( P < 0. 01). Compared with Control-siRNA group,the p53 and cleaved PARP showed up-regulated expression,while Bcl-2 showed down-regulated expression. Conclusion: lncRNA CCAT2 was over-expressed in colon carcinoma cell lines. siRNA-mediated CCAT2 knockdown inhibits the cell proliferations and promotes apoptosis of colon cancer cells in vitro,possibly by activating apoptosis signaling pathway.