本文選題：ELP3 + 肺癌細(xì)胞A549　； 參考：《內(nèi)蒙古大學(xué)》2015年碩士論文

【摘要】：DNA甲基化和組蛋白修飾等表觀遺傳調(diào)節(jié)對基因表達(dá)調(diào)控有重要作用。研究表明,腫瘤形成過程中,表觀遺傳修飾的改變導(dǎo)致基因表達(dá)異常,細(xì)胞發(fā)生癌變。延伸復(fù)合體3(ELP3)是一種與轉(zhuǎn)錄過程密切相關(guān)的組蛋白乙�；D(zhuǎn)移酶,具有鐵硫簇結(jié)構(gòu)域(SAM),對DNA去甲基化具有重要作用,但ELP3是如何調(diào)節(jié)肺癌細(xì)胞的細(xì)胞特性和惡性表型的作用還不清楚。本研究檢測了ELP3在肺癌細(xì)胞A549的表達(dá)定位,并通過基因敲減研究了ELP3對肺癌細(xì)胞的影響及作用機(jī)制。本研究首先利用實(shí)時(shí)定量PCR檢測了不同肺癌細(xì)胞系中ELP3 mRNA水平的表達(dá),結(jié)果表明,肺癌細(xì)胞A549、H460、SK-MES-1的ELP3 mRNA表達(dá)水平均高于肺上皮細(xì)胞,肺癌細(xì)胞系A(chǔ)549的表達(dá)水平最高,故選擇A549做后續(xù)實(shí)驗(yàn)。其次,通過構(gòu)建pGPU6/GFP/Neo siRNA表達(dá)載體,利用脂質(zhì)體法轉(zhuǎn)染A549細(xì)胞,經(jīng)G418篩選,Real-time PCR檢測表明轉(zhuǎn)染細(xì)胞中ELP3 mRNA的表達(dá)與對照組相比降低了85.4%(p0.01),說明ELP3的表達(dá)受到抑制。之后,利用MTT、Traswell小室和軟瓊脂集落實(shí)驗(yàn)對ELP3敲減后癌細(xì)胞的惡性表型進(jìn)行了檢測。MTT結(jié)果表明,與對照細(xì)胞相比,ELP3敲減后細(xì)胞增殖能力下降了24.61%(p0.05)；Traswell小室和軟瓊脂集落結(jié)果表明,細(xì)胞遷移能力下降了52.38%(p0.01),侵襲能力下降了37.90%(p0.05)；軟瓊脂集落實(shí)驗(yàn)表明,錨定非依賴生長能力顯著降低了83.57%(p0.01)。為進(jìn)一步研究ELP3可能的作用機(jī)理,本研究檢測了ELP3敲減后對相關(guān)基因表達(dá)及相應(yīng)的基因啟動(dòng)子區(qū)表觀修飾的影響。實(shí)時(shí)定量PCR結(jié)果表明,ELP3基因被敲減后,BCL2、C-erbA、C-JUN、C-myc、N-myc的mRNA表達(dá)顯著下調(diào),KLF4的mRNA的表達(dá)水平上調(diào),K-ras、SOX2的轉(zhuǎn)錄水平未受影響。亞硫酸氫鹽PCR測序結(jié)果顯示,與對照組相比,BCL2啟動(dòng)子區(qū)DNA甲基化水平提高了5.00%, K-ras啟動(dòng)子區(qū)甲基化水平提高了8.46%。α衛(wèi)星序列(alpha satellit e)甲基化水平降低了10%,微衛(wèi)星序列32的長末端重復(fù)序列(retroviral LTR sequence of minisatellite 32)的甲基化水平提高了18.30%。ChIP研究結(jié)果顯示,敲減ELP3基因后,C-myc和SOX2基因啟動(dòng)子區(qū)組蛋白修飾H3K9me3、H3K4me3、 H3K27me3水平顯著降低。綜上所述,ELP3在肺癌細(xì)胞中高表達(dá),ELP3敲減后癌基因的表達(dá)受到抑制,相應(yīng)的癌基因啟動(dòng)子區(qū)的DNA甲基化水平有所提高,另外,敲減ELP3基因后,相關(guān)基因啟動(dòng)子區(qū)組蛋白修飾也發(fā)生改變。表明ELP3在肺癌細(xì)胞具有DNA去甲基化作用,ELP3可能通過影響相關(guān)基因啟動(dòng)子區(qū)DNA甲基化和組蛋白修飾,改變基因表達(dá)水平,從而影響肺癌細(xì)胞的增殖、遷移、侵襲和錨定非依賴生長能力。
[Abstract]:Epigenetic regulation such as DNA methylation and histone modification plays an important role in the regulation of gene expression.The results showed that the epigenetic modification resulted in abnormal gene expression and carcinogenesis during tumor formation.Extension complex 3 (ELP3) is a histone acetyltransferase, which is closely related to transcription process, and has a ferrithione cluster domain, SAMN, which plays an important role in demethylation of DNA.But it is not clear how ELP3 regulates the cellular properties and malignant phenotypes of lung cancer cells.In this study, the expression of ELP3 in lung cancer cell line A549 was detected, and the effect of ELP3 on lung cancer cell line A549 was studied by gene knockdown.In this study, real-time quantitative PCR was used to detect the expression of ELP3 mRNA in different lung cancer cell lines. The results showed that the expression of ELP3 mRNA in lung cancer cell line A549 was higher than that in lung epithelial cell line, and the expression level of ELP3 mRNA in lung cancer cell line A549 was the highest.So choose A549 to do follow-up experiment.Secondly, the pGPU6/GFP/Neo siRNA expression vector was constructed and transfected into A549 cells by liposome method. The results of G418 screening and Real-time PCR detection showed that the expression of ELP3 mRNA in the transfected cells was lower than that in the control group by 85.4p0.01, indicating that the expression of ELP3 was inhibited.After that, the malignant phenotype of cancer cells after ELP3 knockout was detected by MTT taswell chamber and soft Agar colony assay. The results showed that compared with the control cells, the proliferative ability of the cells after ELP3 knockout was decreased by 24.61% Traswell chamber and soft Agar colony assay.The ability of cell migration and invasion decreased by 52.38% and 37.90%, respectively, and the ability of anchoring independent growth was significantly decreased by 83.57% (p0.01) in soft Agar colony assay.In order to further study the possible mechanism of ELP3, the effects of ELP3 knockout on the expression of related genes and the corresponding epidermal modification of gene promoter were investigated.The results of real-time quantitative PCR showed that the expression of C-erbAerbAC-JUNC-JUNC N-myc mRNA was significantly down-regulated after the knockout of ELP3 gene. The expression level of mRNA of KLF4 was up-regulated and the transcription level of K-rassox2 was not affected.The results of PCR sequencing showed that,Compared with the control group, the methylation level of DNA in the BCL2 promoter increased 5.00, the methylation level in the K-ras promoter increased 8.46%. The methylation level of 偽 -satellite sequence alpha satellit decreased by 10%, and the long terminal repeat sequence of microsatellite sequence 32 was retroviral LTR sequence of minisatellite.The methylation level of 32) increased the level of methylation in the 18.30%.ChIP study.After knockout of ELP3 gene, the level of H3K27me3 decreased significantly when the promoter protein of C-myc and SOX2 gene was modified with histone of H3K9me3m3C3K4me3.In conclusion, the overexpression of ELP3 in lung cancer cells was inhibited, and the DNA methylation level in the promoter region of the corresponding oncogene was increased. In addition, after knockout of the ELP3 gene,The histone modification of the promoter region of the related gene also changed.The results suggest that ELP3 has DNA demethylation effect in lung cancer cells. ELP3 may affect the proliferation and migration of lung cancer cells by affecting the DNA methylation and histone modification in the promoter region of lung cancer cells.Invasion and anchoring independent growth capacity.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S857.4

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本文編號(hào)：1745278