DNMT1对目的基因mRNA表达水平影响的初步研究

发布时间：2018-03-20 16:46

  本文选题：人乳头瘤病毒16　切入点：DNA甲基转移酶1　出处：《北京协和医学院》2017年硕士论文　论文类型：学位论文

【摘要】：背景 在HPV相关肿瘤中,高危型HPV感染,是受染细胞发生恶性转化的必要条件。超过一半的HPV相关肿瘤与HPV 16相关。本课题组前期试验证明人类乳头瘤病毒HPV16E6、E7与DNA甲基转移酶(DNMTs)相关,引起6种抑癌基因(SPARC、TFPI2、RRAD、SFRP1、MT1G 和 NMES1)甲基化发生相应的改变,进而引起基因表达改变,最终可能参与细胞的恶性转化过程。DNMTs主要包括DNMT1和DNMT3,前者主要参与甲基化状态的维持,也是非CpG位点从头甲基化所必需,并与甲基化状态的延伸有关;后者包括DNMT3a、DNMT 3b和DNMT3L。DNMT3a和3b是主要的从头甲基化酶。人们认为DNMT3L不直接参与DNA甲基化,但其与DNMT3a和3b相互作用,可能在转录抑制中发挥作用。。前期研究中我们发现,将SiHa细胞株(HPV16阳性)中的E6、E7基因分别沉默后,4种DNMTs的表达水平均有所下降,但下降程度存在明显差异。而将HPV16 E6、E7同时转入原代角质形成细胞后,在细胞永生化的过程中,4种DNMTs的表达水平也存在不同程度的升高。我们据此假设,除了传统认为的不同功能之外,DNMTs在调控目的基因的甲基化状态时,可能有(相对)关键的DNMT存在。目的本研究拟采用SiHa细胞,应用特异性siRNA沉默DNMT1,观察DNMT1和6种抑癌基因的mRNA表达水平以及DNMT1蛋白表达水平的变化。方法应用针对DNMT1的siRNA,沉默DNMT1。应用qPCR比较沉默前后不同时间点DNMT1的mRNA和蛋白表达的变化,以及6种目的基因的mRNA表达水平的变化。结果转染siRNA后,DNMT1 mRNA和蛋白水平表达下调(p0.05)。6种目的基因的mRNA表达上调(p0.05)结论DNMT1对SiHa细胞中的6种目的基因的mRNA表达可能起抑制作用。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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