沉默SIAH2基因抑制人肝癌细胞HepG2裸鼠移植瘤生长实验研究

Inhibitory effect of silencing SIAH2 by shRNA on subcutaneous tumor of human hepatocellular carcinoma cell line HepG2 in nude mice

目的本研究前期实验发现,靶向SIAH2的shRNA载体能显著抑制HepG2细胞SIAH2mRNA和蛋白的表达,并抑制细胞体外增殖。本研究从体内水平验证靶向SIAH2的干扰载体对人肝癌细胞HepG2细胞裸鼠移植瘤生长的抑制作用。方法转染pGenesil-SIAH2的HepG2细胞作为实验组,命名为HepG2-SIAH2;转染空载体pGenesil-1的HepG2细胞作为阴性对照组,命名为HepG2-neo;未转染任何质粒的HepG2细胞作为空白对照组,通过G418筛选出稳定转染细胞株。将15只裸鼠随机分为3组,接种肿瘤细胞后观察裸鼠成瘤情况。4周后测量肿瘤的体积和瘤体质量,绘制移植瘤生长曲线,并用实时荧光定量PCR和蛋白质印迹法检测移植瘤中SIAH2的表达情况。结果稳定转染pGenesil-SIAH2的细胞株构建成功,3组裸鼠接种癌细胞后均有肿瘤形成。与空白对照组和阴性对照组相比,实验组肿瘤生长速度明显减慢,实验组平均瘤体积(261.57±41.141)mm3,明显低于阴性对照组(494.35±93.236)mm3(P=0.015)和空白对照组(418.3±28.576 5)mm3(P=0.012),差异有统计学意义;实验组平均瘤体质量为(0.162±0.02)g,明显低于阴性对照组(0.358±0.12)g(P=0.032)和空白对照组(0.322±0.24)g(P=0.028)。实验组瘤体内SIAH2mRNA相对表达量为0.83±0.35,明显低于阴性对照组2.35±0.96(P=0.003)和空白对照组2.57±0.41(P=0.006),差异有统计学意义。实验组瘤体内SIAH2蛋白表达量为0.72±0.02,明显低于阴性对照组2.61±0.67(P=0.004)和空白对照组2.49±0.91(P=0.007),差异有统计学意义。结论靶向SIAH2的shRNA干扰载体能有效抑制人肝癌裸鼠移植瘤的生长,SIAH2有可能成为肝癌基因治疗新的分子靶。

OBJECTIVE To investigate the inhibitory effects of silencing SIAH2 gene on the growth of subcutaneous tumor of human hepatocellular carcinoma cell line HepG2 in nude mice.METHODS HepG2 cells were divided into3groups:the HepG2 cells in experimental group were transfected with the recombinant pGenesil-SIAH2 vector,the cells in negative control group were transfected with a control vector pGenesil-1,and the cells in normal control group were without any treatment.All the transfected cells were selected through G418.BALB/C nude mice and were randomly divided into three groups,the growth of the transplanted tumor cells in the nude mice was observed.The tumor growth curve,volume and weight were determined 4weeks after the cell inoculation.The expressions of SIAH2 were analyzed by quantitative real-time PCR,western blot.RESULTS SIAH2knock-downed HepG2 cell was established successfully.Tumor formation was all positive in the 3groups of the nude mice inoculated with the tumor cells.Compared with normal control group and negative control group,the tumor growth slowed significantly in experimental group.The mean volume of experimental group[(261.57±41.141)mm3]was significantly lower than those in negative control group[(494.35±93.236)mm3,P=0.015]and normal control group[(418.3±28.576 5)mm3,P=0.012].The mean weight of experimental group[(0.162±0.02)g]was significantly lower than those in negative control group [(0.358±0.12)g,P=0.032]and normal control group[(0.322±0.24)g,P=0.028].Compared with negative control group(2.35±0.96,P=0.003)and normal control group(2.57±0.41,P=0.006),SIAH2 mRNA expression in transplanted tumor tissues(0.83±0.35)was significantly decreased.Compared with negative control group(2.61±0.67,P=0.004)and normal control group(2.49±0.91,P=0.007),SIAH2 protein expression in transplanted tumor tissues(0.72±0.02)was significantly decreased.CONCLUSION shRNA induced down-regulation of SIAH2 effectively inhibits the growth of transplanted hepatocellular carcinoma,suggesting that SIAH2 gene may be an effective target for gene therapy in treating liver cancer.