长链非编码RNA CCAT2在肝癌中的作用及其机制

Role of long noncoding RNA CCAT2 in modulating biological behaviors of liver cancer cells and the underlying mechanism

目的探讨长链非编码RNA CCAT2(LncRNA CCAT2)在肝癌中的作用及机制。方法选取我院2013年1-6月60例肝癌及其癌旁组织,采用实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测其组织中CCAT2的表达,同时培养正常肝细胞L02和人源性肝癌细胞系Hep G、H2P、SMMC和HLE细胞,采用qRT-PCR检测细胞中CCAT2的表达,采用siRNA干扰细胞CCAT2表达后,在24、48、72、96 h用CCK-8检测SMMC和HLE细胞增殖;Transwell实验分析细胞迁移和侵袭情况;采用流式细胞仪分析CCAT2对SMMC和HLE细胞周期分布和凋亡的影响;Western blot检测P53、Bcl-2、Caspase-8蛋白的表达。结果在肝癌组织和肝癌细胞中,CCAT2的表达显著升高(P均<0.05),CCAT2高表达的患者预后和生存率均较低(P均<0.05)。肝癌细胞中低表达的CCAT2能显著抑制肝癌细胞增殖、迁移和侵袭(P均<0.05)。肝癌细胞中低表达的CCAT2也能使肝癌细胞周期停留在G0/G1期并促进细胞凋亡(P均<0.05)。采用siRNA干扰细胞CCAT2表达后,肝癌细胞中P53和Caspase-8蛋白的表达显著升高(P均<0.05),Bcl-2蛋白的表达显著降低(P<0.05)。结论 CCAT2在肝癌组织和细胞系中高表达,siRNA干扰CCAT2表达后能够抑制细胞增殖、迁移、侵袭以及促进肝癌细胞的凋亡,其机制可能通过升高P53和Caspase-8蛋白的表达和降低Bcl-2蛋白的表达有关。

Objective To investigate the role of long noncoding RNA CCAT2 （LncRNA CCAT2） in modulating the proliferation, migration, invasion and apoptosis of liver cancer cells in vitro and explore the underlying mechanism. Methods Quantitative real-time polymerase chain reaction （qRT-PCR） was used to detect the expression of LncRNA CCAT2 in 60 paired liver cancer and adjacent tissue samples collected from Southwest Hospital between January and June 2013 and in normal human liver cell line L02 and liver cancer cell lines HepG, H2P, SMMC and HLE. SMMC and HLE cells transfected with siRNA-CCAT2 were examined for cell proliferation rate at 24, 48, 72 and 96 h after transfection using CCK-8 assay; the cell migration and invasion were detected with Transwell chamber test, and the cell cycle distribution and apoptosis were analyzed with flow cytometry. Western blotting was used to detect the expression levels of P53, Bcl-2 and caspase-8 in the transfected SMMC and HLE cells. Results The expression level of CCAT2 was significantly increased in the liver cancer tissues and cell lines （all P 〈 0. 05 ）. The patients with a high expression level of CCAT2 had a poorer prognosis and a lower overall survival rate （ all P 〈 0.05 ）. In SMMC and HLE cells,CCAT2 knockdown by small interfering RNA resulted in cell cycle arrested in G0/G1 phase （P 〈 0. 05 ）, promoted the cell apoptosis （ P 〈 0.05 ） , and enhanced the expression levels of P53 and Caspase-8 but decreased Bcl-2 expression （P 〈 0. 05 ）. Conclusion LncRNA CCAT2 is over-expressed in liver cancer tissue and cell lines, siRNA-mediated CCAT2 knockdown suppresses the proliferation, invasion and migration and promotes apoptosis of liver cancer cells in vitro possibly by increasing the expression of P53 and caspase- 8 and decreasing the expression of Bcl-2 protein.