沉默HOXA13基因对肝癌HepG_2和QGY-7703细胞恶性表型的影响

Effects of silencing HOXA13 gene on malignant phenotypes of hepatocellular carcinoma HepG_2 and QGY-7703 cells

目的:探讨沉默HOXA13基因对肝癌细胞株HepG2与QGY-7703恶性表型的影响,为肝癌的诊断及治疗提供新的分子靶点。方法:构建HOXA13基因的干扰重组质粒plent-U6-GFP/si-HOXA13,将其稳定转染至HepG2与QGY-7703细胞;采用RT-PCR和Western blotting法鉴定干扰效果;以HOXA13基因沉默细胞为实验组,以转染空质粒的细胞为对照组,采用四甲基偶氮唑蓝(MTT)法、细胞倍增时间测定、平板克隆形成实验和流式细胞术等分析HOXA13基因沉默后HepG2与QGY-7703细胞生长速度、细胞倍增时间、克隆形成能力和细胞周期等的改变。结果:MTT实验,与对照组比较,实验组细胞的生长速度明显下降;其细胞周期也发生改变,G1期细胞增多、S期细胞减少。对照组HepG2和QGY-7703细胞平均倍增时间分别为(4.59±0.27)和(4.93±0.17)h,实验组HepG2和QGY-7703细胞平均倍增时间分别为(6.02±0.86)和(6.43±0.66)h,2组间比较差异有统计学意义(P<0.05)。对照组HepG2和QGY-7703细胞平均细胞克隆形成数分别为(264.00±12.62)和(269.00±4.55)个,实验组HepG2和QGY-7703细胞平均细胞克隆形成数目分别为(165.00±10.61)和(215.00±4.43)个,2组比较差异有统计学意义(P<0.01)。结论:HOXA13基因可以使肝癌细胞HepG2与QGY-7703的增殖加快、克隆形成能力增强、G1期细胞减少、S期细胞增多,可以成为肝癌诊断和治疗新的分子靶点。

Objective:To investigate the effects of silencing HOXA13 gene on the malignant phenotypes of hepatocellular carcinoma cell lines HepG 2 and QGY-7703,and to provide a new molecular target for the diagnosis and treatment of hepatocellular carcinoma.Methods:The interference recombinant plasmid of plent-U6-GFP/si-HOXA13 was constructed,then it was stably transfected into the HepG 2 and QGY-7703 cells.The interfering effects of HOXA13 gene were determined by RT-PCR and Western blotting methods.The HepG 2 and QGY-7703 cells transfected with HOXA13 knockdown were used as experimental group,and the HepG 2 and QGY-7703 cells transfected with empty plasmid were used as control group.Then the growth speed was examined by MTT assay,the cell doubling time was examined by double time assay,the colony formation ability was examined by colony formation assay,and the cell cycle was examined by flow cytometry.Results:The MTT assay results showed that compared with control group,the growth speeds of HepG 2 and GY-7703 cells in experimental group were significantly decreased and the cell cycles were changed;the number of HepG 2 and QGY-7703 cells in G 1 phase was increased and the number of cells in S phase was decreased.The doubling time of HepG 2 and QGY-7703 cells in control group and experimental group were(4.59±0.27),(4.93±0.17),(6.02±0.86),and(6.43±0.66)h,and the differences between control group and experimental group were significant(P<0.05).The colony number of HepG 2 and QGY-7703 cells in control group and experimental group were 264.00±12.62,269.00±4.55,165.00±10.61,and 215.00±4.43,and the differences between control group and experimental group were significant(P<0.01).Conclusion:HOXA13 can increase the proliferation,enhance the clone formation,decrease the number of cells at G 1 phase and increase the number of cells at S phase in the hepatocellular carcinoma HepG 2 and QGY-7703 cells;and it may used as a new molecular target for diagnosis and treatment of hepatocellular carcinoma.