沉默SIAH2基因抑制人肝癌细胞HepG2裸鼠移植瘤生长实验研究

发布时间：2018-10-23 14:57

【摘要】：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。
[Abstract]:Aim in the previous study, it was found that the shRNA vector targeting SIAH2 could significantly inhibit the expression of SIAH2mRNA and protein in HepG2 cells and inhibit the proliferation of HepG2 cells in vitro. The aim of this study was to investigate the inhibitory effect of the interference vector targeting SIAH2 on the growth of human hepatoma HepG2 cells in nude mice in vivo. Methods HepG2 cells transfected with pGenesil-SIAH2 were used as experimental group, HepG2 cells named as HepG2-SIAH2; transfected empty vector pGenesil-1 as negative control group and HepG2 cells named HepG2-neo; as blank control group. Stable transfection cell lines were screened by G418. Fifteen nude mice were randomly divided into 3 groups. The tumorigenesis of nude mice was observed after inoculation of tumor cells, the tumor volume and tumor mass were measured 4 weeks later, and the growth curve of transplanted tumor was drawn. The expression of SIAH2 in transplanted tumor was detected by real-time fluorescence quantitative PCR and Western blotting. Results the cell lines transfected with pGenesil-SIAH2 stably were successfully constructed and tumor formation was found in all the 3 groups of nude mice inoculated with cancer cells. Compared with the blank control group and the negative control group, the tumor growth rate in the experimental group was significantly slower. The mean tumor volume (261.57 卤41.141) mm3, in the experimental group was significantly lower than that in the negative control group (494.35 卤93.236) mm3 (P0. 015) and the blank control group (418.3 卤28.576 5) mm3 (P0. 012). The mean tumor mass in the experimental group was (0.162 卤0.02g), which was significantly lower than that in the negative control group (0.358 卤0.12) g (P0. 032) and the blank control group (0.322 卤0. 24) g (P0. 028). The relative expression of SIAH2mRNA in the experimental group was 0.83 卤0.35, which was significantly lower than that in the negative control group (2.35 卤0.96) and the blank control group (2.57 卤0.41) (P0. 006). The expression of SIAH2 protein in the experimental group was 0.72 卤0.02, which was significantly lower than that in the negative control group (2.61 卤0.67) and the blank control group (2.49 卤0.91) (P0. 007). Conclusion shRNA interference vector targeting SIAH2 can effectively inhibit the growth of human hepatoma xenografts in nude mice. SIAH2 may be a new molecular target for gene therapy of hepatocellular carcinoma.
【作者单位】：赣南医学院第一附属医院消化内科;赣南医学院第一附属医院医务科;
【基金】：国家自然科学基金(81160257)
【分类号】：R735.7

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