本文關(guān)鍵詞： siM2PK-1 CCND1 RNA干擾 DNA甲基化 缺氧誘導 微小RNA HepG2細胞 基因表達　出處：《安徽醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：目的:研究siM2PK-1分子對CCND1表達的抑制作用并探討其機制。方法:通過克隆形成實驗及流式細胞術(shù)檢測siM2PK-1分子對于肝癌細胞增殖與周期的影響,利用實時定量PCR和Western blot檢測siM2PK-1分子對于CCND1的表達的影響,過表達PKM2及利用其它針對PKM2的干涉實驗確定siM2PK-1對于CCND1抑制作用的特異性,生物信息學分析及報告基因檢測初步探究了siM2PK-1抑制CCND1表達的機制。結(jié)果:克隆形成實驗發(fā)現(xiàn)siM2PK-1分子對于SK-Hep-1細胞具有抑制增殖的作用,流式細胞術(shù)檢測發(fā)現(xiàn)siM2PK-1可以引起細胞周期阻滯于G0-G1期,實時定量PCR和Western blot證實siM2PK-1分子對于CCND1的表達在mRNA水平和蛋白水平都有影響,過表達PKM2分子不能引起CCND1分子在mRNA和蛋白水平的上調(diào)表達,而利用其他的針對PKM2分子的siRNA也不能使CCND1的表達下調(diào),進而得出siM2PK-1分子具有雙靶向作用,而進一步利用生物信息學分析結(jié)合報告基因確定siM2PK-1可能通過不完全互補的模式與CCND1分子啟動子區(qū)的序列結(jié)合進而引起其啟動子區(qū)的CpG島的甲基化。結(jié)論:抑制PKM2的siM2PK-1分子具有協(xié)同抑制CCND1分子表達的作用。 目的:探討不同缺氧條件對HepG2細胞中miR-210表達水平的影響。方法:采用CoCl_2、物理缺氧以及Na_2SO_3三種不同的缺氧條件誘導HepG2細胞,利用實時定量PCR技術(shù)檢測不同缺氧模式誘導前后HepG2細胞miR-210等表達變化。結(jié)果:三種缺氧模式均可以誘導HepG2細胞miR-210表達上調(diào),其表達與CoCl_2及Na_2SO_3的濃度有劑量依賴關(guān)系。結(jié)論:CoCl_2、物理缺氧以及Na_2SO_3三種不同的缺氧條件均可以有效誘導HepG2細胞中miR-210的表達上調(diào),三種模型均可用于缺氧誘導miRNA研究,同時進一步證明miR-210表達上調(diào)是由于細胞缺氧所致。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346

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