本文選題：人乳頭瘤病毒16　切入點(diǎn)：DNA甲基轉(zhuǎn)移酶1　出處：《北京協(xié)和醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景 在HPV相關(guān)腫瘤中,高危型HPV感染,是受染細(xì)胞發(fā)生惡性轉(zhuǎn)化的必要條件。超過一半的HPV相關(guān)腫瘤與HPV 16相關(guān)。本課題組前期試驗(yàn)證明人類乳頭瘤病毒HPV16E6、E7與DNA甲基轉(zhuǎn)移酶(DNMTs)相關(guān),引起6種抑癌基因(SPARC、TFPI2、RRAD、SFRP1、MT1G 和 NMES1)甲基化發(fā)生相應(yīng)的改變,進(jìn)而引起基因表達(dá)改變,最終可能參與細(xì)胞的惡性轉(zhuǎn)化過程。DNMTs主要包括DNMT1和DNMT3,前者主要參與甲基化狀態(tài)的維持,也是非CpG位點(diǎn)從頭甲基化所必需,并與甲基化狀態(tài)的延伸有關(guān);后者包括DNMT3a、DNMT 3b和DNMT3L。DNMT3a和3b是主要的從頭甲基化酶。人們認(rèn)為DNMT3L不直接參與DNA甲基化,但其與DNMT3a和3b相互作用,可能在轉(zhuǎn)錄抑制中發(fā)揮作用。。前期研究中我們發(fā)現(xiàn),將SiHa細(xì)胞株(HPV16陽性)中的E6、E7基因分別沉默后,4種DNMTs的表達(dá)水平均有所下降,但下降程度存在明顯差異。而將HPV16 E6、E7同時轉(zhuǎn)入原代角質(zhì)形成細(xì)胞后,在細(xì)胞永生化的過程中,4種DNMTs的表達(dá)水平也存在不同程度的升高。我們據(jù)此假設(shè),除了傳統(tǒng)認(rèn)為的不同功能之外,DNMTs在調(diào)控目的基因的甲基化狀態(tài)時,可能有(相對)關(guān)鍵的DNMT存在。目的本研究擬采用SiHa細(xì)胞,應(yīng)用特異性siRNA沉默DNMT1,觀察DNMT1和6種抑癌基因的mRNA表達(dá)水平以及DNMT1蛋白表達(dá)水平的變化。方法應(yīng)用針對DNMT1的siRNA,沉默DNMT1。應(yīng)用qPCR比較沉默前后不同時間點(diǎn)DNMT1的mRNA和蛋白表達(dá)的變化,以及6種目的基因的mRNA表達(dá)水平的變化。結(jié)果轉(zhuǎn)染siRNA后,DNMT1 mRNA和蛋白水平表達(dá)下調(diào)(p0.05)。6種目的基因的mRNA表達(dá)上調(diào)(p0.05)結(jié)論DNMT1對SiHa細(xì)胞中的6種目的基因的mRNA表達(dá)可能起抑制作用。
[Abstract]:Background in HPV associated tumors, high-risk HPV infection, More than half of the tumors associated with HPV are associated with HPV16. Our previous studies have demonstrated that HPV16 E6E7 is associated with DNA methyltransferase (DNMTs). The methylation of six tumor suppressor genes, SPARCU TFPI2, RRADG and NMES1), may result in the change of gene expression, which may be involved in the malignant transformation process of cells. DNMTs mainly include DNMT1 and DNMT3, the former is mainly involved in the maintenance of methylation state, the former is involved in the maintenance of the methylation state, and the other is involved in the maintenance of the methylation state. It is also necessary for non-#en0# site ab initio methylation and is related to the extension of the methylation state; the latter includes DNMT3aAN DNMT3b and DNMT3L.DNMT3a and 3b are the main ab initio methylases. It is believed that DNMT3L is not directly involved in DNA methylation, but it interacts with DNMT3a and 3b. It may play a role in transcription inhibition... In previous studies, we found that the expression levels of E6 / E7 gene in SiHa cell line HPV16 were decreased after silencing respectively. However, there was significant difference in the degree of decrease. However, after HPV16 E6 E7 was transferred into primary keratinocytes at the same time, the expression levels of four DNMTs increased in different degrees during the immortalization of the cells. In addition to the conventional belief that DNMTs have different functions, there may be (relatively) critical DNMT in regulating the methylation status of the target gene. Specific siRNA silencing DNMT1 was used to observe the changes of mRNA expression level and DNMT1 protein expression level of DNMT1 and 6 tumor suppressor genes. Methods the changes of mRNA and protein expression of DNMT1 at different time points before and after DNMT1 silencing were compared with qPCR. Results after transfection of siRNA, the expression of DNMT1 mRNA and protein down-regulated the mRNA expression of p0.05n.6 target genes. Conclusion DNMT1 may inhibit the mRNA expression of 6 target genes in SiHa cells.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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相關(guān)期刊論文 前1條

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