【作者】 刘红霞；

【导师】 郭占军；

【作者基本信息】 河北医科大学 ， 内科学（专业学位）， 2017， 硕士

【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P<0.05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(P<0.05);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:P<0.05;第14天:P<0.05;21天:P<0.05)。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。更多还原

【Abstract】 Objective: The lysine methyltransferase SET8,which is mainly involved in gene transcription,DNA synthesis and repair,heterochromatin formation,genome stability,cell cycle process and other aspects of regulation.As such,it is predicted that SET8 might be involved in the development and progression of multiple tumors.Our previous study suggested that the lysine methyltransferase SET8,whose expression was regulated by miR-502 through the binding site in the 3’UTR of SET8,implicated in the prognosis of hepatocellular carcinoma.Subsequent studies have shown that siRNA-mediated silencing of the SET8 gene could significantly inhibit cell proliferation,invasion and migration abilities for human hepatocellular carcinoma of SMMC-7721 cells in vitro.This study is to investigate the effect of small interference siRNAmediated silencing of the SET8 gene on growth of human hepatocellular carcinoma SMMC-7721 cells in vivo.Methods:1 Designed and transcriptive synthesized a fluorescein-labeled siRNAs targeting the SET8 gene in vitro,which were transfected into SMMC-7721 cells by LipofectamineTM2000.2 SET8-siRNA and control-siRNA stably transfected SMMC-7721 monoclonal cells was selected by puromycin.3 Western Blotting techniques were used to verification the inhibitory effect of siRNA on the expressions of SET8 protein.4 The animal experiments were used to observe the effect of SET8 gene silencing on hepatocellular carcinoma.The SET8-siRNA stably transfected and control-siRNA stably transfected SMMC-7721 cells were subcutaneously inoculated into the subcutaneous of the mice.The tumor size and growth rate of nude mice were observed every week,and the tumor volume was calculated.The green fluorescent images for the xenograft was measured using the NightOwl Bioimager(Berthold Technologies,Bad Wildbad,Germany)once a week.5 SPSS 21.0 statistical software was used for statistical analysis,t test and One-way ANOVA were used to compare differences between groups,with P <0.05 was considered statistically significant.Results:1 The cells transfected with SET8-siRNA and control-si RNA were successfully constructed.The transfection efficiency was 100% under fluorescence microscope scan.2 Compared with control-siRNA group and blank-control group,the expression of SET8 protein in SET8-siRNA group was significantly reduced(P<0.05),and the inhibition rate was about 49% compared with controlsiRNA group.3 Compared with control-siRNA group and blank-control group,the growth of xenografts in SET8-siRNA group was significantly inhibited on the7 th,14th and 21 st day after implantation(the 7th day,P<0.05;the 14 th day,P<0.05;the 21 st day,P<0.05).Conclusion:SiRNA-mediated silencing of the SET8 gene could inhibit the growth of hepatocellular carcinoma in vivo.更多还原