SET8基因沉默抑制肝细胞肝癌的研究

发布时间：2018-03-10 21:22

本文选题：肝细胞肝癌　切入点：SET8　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:SET8是一种赖氨酸甲基转移酶,主要参与基因转录,DNA合成与修复,异染色质形成,基因组的稳定性,细胞周期进程等方面的调控;另有多项研究显示SET8可能与多种肿瘤的发生和发展密切相关。我们的前期研究发现mi R-502可调控SET8基因的表达,并且发现SET8的表达量与肝细胞肝癌预后相关;随后在体外实验中,利用小干扰RNA(siRNA)将SMMC-7721细胞中的SET8基因表达沉默,发现SET8基因沉默可显著抑制肝细胞肝癌细胞在体外的迁移、增殖和侵袭能力,因此,本实验同样利用siRNA沉默SET8基因的表达,探讨其对肝癌SMMC-7721细胞在裸鼠体内生长的影响。方法:1设计并合成用于人SET8基因沉默表达的siRNA(SET8-siRNA)及对照的control-si RNA;并采用荧光标记法标记SET8-siRNA及control-siRNA,利用脂质体(Lipofectamine TM2000)将两种siRNA转染入人肝癌SMMC-7721细胞内。2嘌呤霉素筛选稳定表达SET8-siRNA及control-siRNA的SMMC-7721细胞株。3利用Western blot检测siRNA对SET8基因沉默表达作用。4裸鼠荷瘤实验观察SET8基因沉默对肝细胞肝癌的作用:将稳定转染SET8-siRNA及control-si RNA细胞株分别种植到裸鼠皮下,每周观察裸鼠体内肿瘤的大小及生长速度,计算瘤体体积,并利用小动物活体成像系统进行观察。5采用SPSS 21.0统计学软件进行统计分析,t检验和单因素方差分析(One-way ANOVA)比较组间差异,以P0.05表示差异有统计学意义。结果:1成功构建SET8-siRNA及control-siRNA稳定转染的细胞株,荧光显微镜下可观察转染率为100%。2与control-siRNA组及blank-control组相比,SET8-siRNA组SET8蛋白表达量受到显著抑制(P0.05);与control-siRNA组相比抑制率高达约49%。3与control-siRNA组及blank-control组比较,SET8-siRNA组于裸鼠体内异种种植的瘤体在种植后第7、14、21天生长受到显著抑制(第7天:P0.05;第14天:P0.05;21天:P0.05)。结论:体内实验发现SiRNA介导的SET8基因沉默可以抑制肝细胞肝癌的生长。
[Abstract]:Objective to investigate the role of 1% SET8 in the synthesis and repair of gene transcription DNA, the formation of heterochromatin, the stability of genome, the process of cell cycle and so on. Several other studies have shown that SET8 may be closely related to the occurrence and development of various tumors. Our previous studies have found that miR-502 regulates the expression of SET8 gene, and that the expression of SET8 is related to the prognosis of hepatocellular carcinoma. The expression of SET8 gene in SMMC-7721 cells was silenced by small interfering RNAs. It was found that SET8 gene silencing could significantly inhibit the migration, proliferation and invasion of hepatocellular carcinoma cells in vitro. Therefore, siRNA was also used to silence the expression of SET8 gene. To investigate its effect on the growth of hepatocellular carcinoma (SMMC-7721) cells in nude mice. Methods: 1 designed and synthesized siRNA-SET8-siRNAs for silencing expression of human SET8 gene and control-siRNA as control, and labeled SET8-siRNA and control-siRNAs with fluorescent labeling method, and used liposome Lipofectamine TM2000 to label the two kinds of siRNA. Screening of SMMC-7721 Cell Lines with stable expression of SET8-siRNA and control-siRNA by transfection into Human Hepatocellular carcinoma SMMC-7721 Cell Lines. 4 the effect of SET8 Gene silencing on Hepatocellular carcinoma in Nude mice by detecting the silencing expression of SET8 Gene by Western blot. 4. The effect of SET8 Gene silencing on Hepatocellular carcinoma in Nude mice. Stable transfection of SET8-siRNA and control-si RNA cells were implanted into nude mice subcutaneously. The tumor size and growth rate in nude mice were observed weekly, and the tumor volume was calculated. The small animal living imaging system was used to observe the differences between the two groups using SPSS 21.0 statistical software for statistical analysis and One-way ANOVA (One-way ANOVA). Results the stable transfected SET8-siRNA and control-siRNA cell lines were successfully constructed by 1: 1. The expression of SET8 protein in SET8-siRNA group was significantly inhibited compared with control-siRNA group and blank-control group, and the inhibition rate was about 49.3% in control-siRNA group, 49.3% in control-siRNA group and blank-control group in blank-control group. The growth of the body was significantly inhibited at the 21st day after planting (7 days: P0.05; 14 days: P0.05; 21 days: P0.05.Conclusion: SiRNA mediated SET8 gene silencing can inhibit the growth of hepatocellular carcinoma in vivo.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

【参考文献】