本文關(guān)鍵詞： siM2PK-1 CCND1 RNA干擾 DNA甲基化 缺氧誘導(dǎo) 微小RNA HepG2細(xì)胞 基因表達(dá)　出處：《安徽醫(yī)科大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:研究siM2PK-1分子對(duì)CCND1表達(dá)的抑制作用并探討其機(jī)制。方法:通過(guò)克隆形成實(shí)驗(yàn)及流式細(xì)胞術(shù)檢測(cè)siM2PK-1分子對(duì)于肝癌細(xì)胞增殖與周期的影響,利用實(shí)時(shí)定量PCR和Western blot檢測(cè)siM2PK-1分子對(duì)于CCND1的表達(dá)的影響,過(guò)表達(dá)PKM2及利用其它針對(duì)PKM2的干涉實(shí)驗(yàn)確定siM2PK-1對(duì)于CCND1抑制作用的特異性,生物信息學(xué)分析及報(bào)告基因檢測(cè)初步探究了siM2PK-1抑制CCND1表達(dá)的機(jī)制。結(jié)果:克隆形成實(shí)驗(yàn)發(fā)現(xiàn)siM2PK-1分子對(duì)于SK-Hep-1細(xì)胞具有抑制增殖的作用,流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)siM2PK-1可以引起細(xì)胞周期阻滯于G0-G1期,實(shí)時(shí)定量PCR和Western blot證實(shí)siM2PK-1分子對(duì)于CCND1的表達(dá)在mRNA水平和蛋白水平都有影響,過(guò)表達(dá)PKM2分子不能引起CCND1分子在mRNA和蛋白水平的上調(diào)表達(dá),而利用其他的針對(duì)PKM2分子的siRNA也不能使CCND1的表達(dá)下調(diào),進(jìn)而得出siM2PK-1分子具有雙靶向作用,而進(jìn)一步利用生物信息學(xué)分析結(jié)合報(bào)告基因確定siM2PK-1可能通過(guò)不完全互補(bǔ)的模式與CCND1分子啟動(dòng)子區(qū)的序列結(jié)合進(jìn)而引起其啟動(dòng)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(dá)的作用。 目的:探討不同缺氧條件對(duì)HepG2細(xì)胞中miR-210表達(dá)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(dǎo)HepG2細(xì)胞,利用實(shí)時(shí)定量PCR技術(shù)檢測(cè)不同缺氧模式誘導(dǎo)前后HepG2細(xì)胞miR-210等表達(dá)變化。結(jié)果:三種缺氧模式均可以誘導(dǎo)HepG2細(xì)胞miR-210表達(dá)上調(diào),其表達(dá)與CoCl_2及Na_2SO_3的濃度有劑量依賴(lài)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(dǎo)HepG2細(xì)胞中miR-210的表達(dá)上調(diào),三種模型均可用于缺氧誘導(dǎo)miRNA研究,同時(shí)進(jìn)一步證明miR-210表達(dá)上調(diào)是由于細(xì)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(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R346

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 倪勤;RNA干擾技術(shù)防治病毒性肝炎的研究[J];國(guó)外醫(yī)學(xué).流行病學(xué).傳染病學(xué)分冊(cè);2003年04期

2 李光明,謝青,史毅,李定國(guó),金由辛;siRNA對(duì)HSC結(jié)締組織生長(zhǎng)因子的抑制作用[J];上海第二醫(yī)科大學(xué)學(xué)報(bào);2005年02期

3 燕春艷;RNA干擾技術(shù)研究進(jìn)展[J];實(shí)用醫(yī)藥雜志;2003年09期

4 馬鵬鵬,葛曄華,郭睿,馬靜,陳平,薛社普,韓代書(shū);小分子干擾RNA(siRNA)表達(dá)載體沉默靶基因的研究[J];解剖學(xué)報(bào);2004年06期

5 郭述良,羅永艾;RNA干擾及其在呼吸系統(tǒng)疾病研究中的應(yīng)用[J];國(guó)外醫(yī)學(xué).內(nèi)科學(xué)分冊(cè);2004年12期

6 趙洪波,張嘉寧;RNA干擾技術(shù)的應(yīng)用[J];大連醫(yī)科大學(xué)學(xué)報(bào);2003年04期

7 范怡敏,耿飛,吳興中;RNAi的機(jī)制及RNAi技術(shù)的應(yīng)用[J];醫(yī)學(xué)綜述;2004年04期

8 王占黎,盧景芬,張亮仁,張禮和;RNA干擾與藥物的研究進(jìn)展[J];國(guó)外醫(yī)學(xué).藥學(xué)分冊(cè);2004年04期

9 熊潔,陳寶安;RNA干擾技術(shù)在白血病研究中的應(yīng)用前景[J];國(guó)外醫(yī)學(xué).腫瘤學(xué)分冊(cè);2004年11期

10 胡曉霞,李力;RNA干擾技術(shù)及在婦科腫瘤研究中的應(yīng)用[J];國(guó)外醫(yī)學(xué).婦產(chǎn)科學(xué)分冊(cè);2005年02期

相關(guān)會(huì)議論文 前10條

1 鄧清華;劉國(guó)文;劉磊;王建國(guó);朱曉巖;李小兵;王哲;;siRNA特異性抑制犢牛原代肝細(xì)胞SREBP-1c基因的表達(dá)[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)家畜內(nèi)科學(xué)分會(huì)第七屆代表大會(huì)暨學(xué)術(shù)研討會(huì)論文集（上冊(cè)）[C];2011年

2 季愛(ài)民;蘇丹;車(chē)鷗;李文適;孫靚;張忠義;楊彬;;殼聚糖/siRNA納米粒介導(dǎo)的功能性基因沉默研究(英文)[A];2010年中國(guó)藥學(xué)大會(huì)暨第十屆中國(guó)藥師周論文集[C];2010年

3 黃偉;呂明;金明姬;楊長(zhǎng)青;高鐘鎬;;mPEG-PCL-g-PEI聚合物的合成及其siRNA遞送性能[A];2010年中國(guó)藥學(xué)大會(huì)暨第十屆中國(guó)藥師周論文集[C];2010年

4 陳宇;鄭海濤;楊力建;陳少華;沈琦;劉思初;李，

本文編號(hào)：1481926