抑制PKM2的siRNA协同抑制CCND1的作用机制研究

发布时间：2018-02-01 13:05

本文关键词： siM2PK-1 CCND1 RNA干扰 DNA甲基化缺氧诱导微小RNA HepG2细胞基因表达　出处：《安徽医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:研究siM2PK-1分子对CCND1表达的抑制作用并探讨其机制。方法:通过克隆形成实验及流式细胞术检测siM2PK-1分子对于肝癌细胞增殖与周期的影响,利用实时定量PCR和Western blot检测siM2PK-1分子对于CCND1的表达的影响,过表达PKM2及利用其它针对PKM2的干涉实验确定siM2PK-1对于CCND1抑制作用的特异性,生物信息学分析及报告基因检测初步探究了siM2PK-1抑制CCND1表达的机制。结果:克隆形成实验发现siM2PK-1分子对于SK-Hep-1细胞具有抑制增殖的作用,流式细胞术检测发现siM2PK-1可以引起细胞周期阻滞于G0-G1期,实时定量PCR和Western blot证实siM2PK-1分子对于CCND1的表达在mRNA水平和蛋白水平都有影响,过表达PKM2分子不能引起CCND1分子在mRNA和蛋白水平的上调表达,而利用其他的针对PKM2分子的siRNA也不能使CCND1的表达下调,进而得出siM2PK-1分子具有双靶向作用,而进一步利用生物信息学分析结合报告基因确定siM2PK-1可能通过不完全互补的模式与CCND1分子启动子区的序列结合进而引起其启动子区的CpG岛的甲基化。结论:抑制PKM2的siM2PK-1分子具有协同抑制CCND1分子表达的作用。目的:探讨不同缺氧条件对HepG2细胞中miR-210表达水平的影响。方法:采用CoCl_2、物理缺氧以及Na_2SO_3三种不同的缺氧条件诱导HepG2细胞,利用实时定量PCR技术检测不同缺氧模式诱导前后HepG2细胞miR-210等表达变化。结果:三种缺氧模式均可以诱导HepG2细胞miR-210表达上调,其表达与CoCl_2及Na_2SO_3的浓度有剂量依赖关系。结论:CoCl_2、物理缺氧以及Na_2SO_3三种不同的缺氧条件均可以有效诱导HepG2细胞中miR-210的表达上调,三种模型均可用于缺氧诱导miRNA研究,同时进一步证明miR-210表达上调是由于细胞缺氧所致。
[Abstract]:Objective: to study the inhibitory effect of siM2PK-1 on CCND1 expression and its mechanism. The effects of siM2PK-1 molecules on the proliferation and cell cycle of hepatoma cells were detected by clone formation assay and flow cytometry. The effect of siM2PK-1 on the expression of CCND1 was detected by real-time quantitative PCR and Western blot. Overexpression of PKM2 and other interference experiments aimed at PKM2 were used to determine the specificity of siM2PK-1 in CCND1 inhibition. Bioinformatics analysis and reporter gene detection preliminarily explored the mechanism of siM2PK-1 inhibiting CCND1 expression. Clone formation assay showed that siM2PK-1 molecules could inhibit the proliferation of SK-Hep-1 cells. Flow cytometry showed that siM2PK-1 could cause cell cycle arrest in G0-G1 phase. Real-time quantitative PCR and Western blot confirmed that siM2PK-1 molecules had an effect on the expression of CCND1 at both mRNA and protein levels. Overexpression of PKM2 could not induce the up-regulation of CCND1 at the level of mRNA and protein. The expression of CCND1 could not be down-regulated by using other siRNA targeting PKM2 molecules, and it was concluded that the siM2PK-1 molecule had a double targeting effect. Further use of bioinformatics analysis combined with the reporter gene to determine that siM2PK-1 may bind to the sequence of the promoter region of CCND1 through incomplete complementary patterns, thus causing the CP of its promoter region. Methylation of G Island. Conclusion:. SiM2PK-1 molecules that inhibit the expression of PKM2 have synergistic effect on the expression of CCND1 molecules. Objective: to investigate the effect of hypoxia on the expression of miR-210 in HepG2 cells. Methods: CoCl_2 was used. Three different hypoxia conditions, physical hypoxia and Na_2SO_3, induced HepG2 cells. Real time quantitative PCR technique was used to detect the changes of miR-210 expression in HepG2 cells before and after hypoxia induction. All three hypoxia patterns could induce up-regulation of miR-210 expression in HepG2 cells. There is a dose-dependent relationship between the expression of CoCl_2 and Na_2SO_3. Conclusion: CoCl2. Three different hypoxia conditions, physical hypoxia and Na_2SO_3, could effectively induce the up-regulation of miR-210 expression in HepG2 cells. All three models can be used in the study of hypoxia induced miRNA, and it is further proved that the up-regulation of miR-210 expression is caused by anoxia.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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