NCAPH在宫颈癌发生发展中的作用及其机制的初步研究

发布时间：2018-05-05 06:11

本文选题：宫颈癌 + HPVE7　；参考：《山东大学》2017年硕士论文

【摘要】：背景:宫颈癌是全球最常见的妇科肿瘤之一,人乳头瘤病毒(Human papillomavirus,HPV)的持续感染是其首要启动因素。HPV是一种双链环状DNA病毒,其编码的早期蛋白E6、E7是导致宫颈细胞恶性转化的关键蛋白。然而,仅有HPV的感染不能导致细胞的恶性转化,它仅作为宫颈致癌过程的第一次打击。HPV感染宫颈上皮后所诱导的宿主细胞内一系列的分子信号通路的改变,最终导致细胞发生了恶性转化。然而,目前HPV致宫颈癌发生发展的机制仍有待进一步阐明。凝缩蛋白Ⅰ复合体的非染色体结构维持蛋白的亚单位H(Non-SMC CondensinⅠ Complex SubunitH,NCAPH)基因,主要参与间期核染色质转变成高度螺旋的核染色体,以往有研究显示:NCAPH与非小细胞肺癌的不良预后有关,这提示NCAPH可能与肿瘤的发生发展有关,但机制不明。目的:本课题拟通过体外实验初步阐明NCAPH在宫颈癌中的表达情况、对宫颈癌细胞生长的影响及其可能机制,初步揭示HPVE7与NCAPH之间的调控关系。方法:1)收集10例正常宫颈及22例宫颈癌的新鲜组织标本,提取组织内的总RNA并逆转录为cDNA,同法提取三种宫颈癌细胞系中的总RNA并逆转录为cDNA,实时荧光定量PCR法检测NCAPH的表达,对比其在正常宫颈及宫颈癌中的表达差异。收集65例正常宫颈组织及149例宫颈癌的石蜡标本,免疫组织化学方法检测其中NCAPH蛋白的表达,对比正常宫颈及宫颈癌中NCAPH蛋白的表达差异;2)培养宫颈癌细胞系HeLa和SiHa,瞬时转染靶向NCAPH siRNA,利用CCK-8试剂盒、平板克隆形成实验检测NCAPH对细胞增殖的影响;利用transwell迁移与侵袭实验检测NCAPH对细胞迁移与侵袭能力的影响;3)培养HeLa和SiHa,转染NCAPH siRNA,利用Western Blot和实时荧光定量PCR法检测相关蛋白的表达变化;4)利用JASPAR及PROMO软件预测NCAPH启动子区的转录因子,挑选评分最高的转录因子E2F1;在HeLa和SiHa中干扰和过表达转录因子E2F1,检测NCAPH的mRNA和蛋白表达变化;5)将NCAPH基因启动子区序列连接到pGL3-Basic载体上,构建转录因子E2F1的过表达载体,然后共转染293T细胞,利用双荧光素酶报告检测系统验证转录因子E2F1是否可以结合NCAPH启动子区,激活NCAPH基因的转录;6)培养HPV16E7高表达的细胞系RPE1 E7及其对照细胞系RPE1,检测E7过表达对NCAPH及转录因子E2F1的表达的影响;在HeLa和SiHa中干扰E7,检测NCAPH及转录因子E2F1的表达变化;在RPE1E7细胞中干扰转录因子E2F1的表达,检测敲低E2F1能否影响E7对NCAPH表达的影响。结果:1)NCAPH在3种宫颈癌细胞系中均有表达;与正常宫颈组织相比,NCAPH在宫颈癌组织中的表达水平显著升高;2)CCK-8、平板克隆形成实验结果显示:干扰宫颈癌细胞中NCAPH的表达后,细胞增殖能力明显下降,克隆形成能力显著降低;3)干扰宫颈癌细胞中NCAPH的表达后,细胞的迁移和侵袭能力均受到明显抑制,上皮相关的蛋白ZO-1表达水平显著升高,而间叶相关的蛋白表达水平Vimentin、Snail显著下降;4)干扰宫颈癌细胞中NCAPH的表达后,细胞内的总AKT没有明显变化,而丝氨酸473位点磷酸化的AKT蛋白的表达显著降低;5)转录因子E2F1可以结合在NCAPH的启动子区并上调NCAPH的转录,HPV的关键致癌蛋白E7可以通过调控E2F1上调其下游的NCAPH的表达。结论:NCAPH可能作为一种新的癌基因参与宫颈癌的发生发展。它可以显著促进宫颈癌细胞的增殖,并通过促进EMT增强宫颈癌细胞的迁移和侵袭能力。HPV E7可能通过转录因子E2F1激活NCAPH的转录从而参与宫颈致癌过程。
[Abstract]:Background: cervical cancer is one of the most common gynecologic tumors in the world. The persistent infection of Human papillomavirus (HPV) is its primary factor..HPV is a double chain ring DNA virus, which encodes early protein E6. E7 is the key protein that causes malignant transformation of cervix cells. However, the infection only of HPV can not lead to cells. Malignant transformation, it is only a series of changes in the molecular signaling pathway in the host cell induced by.HPV infection of the cervix epithelium after the first carcinogenic process of the cervix, which eventually leads to the malignant transformation of the cells. However, the mechanism of the occurrence and development of cervical cancer in HPV still remains to be clarified. The subunit H (Non-SMC Condensin I Complex SubunitH, NCAPH), a subunit of chromosome structure maintaining protein, mainly participates in the transformation of the interphase nucleus chromatin into a highly spiral nuclear chromosome. Previous studies have shown that NCAPH is associated with the poor prognosis of non-small cell lung cancer, which suggests that NCAPH may be associated with the development of tumor, but the mechanism is unknown. The objective of this study is to preliminarily clarify the expression of NCAPH in cervical cancer and the possible mechanism of cervical cancer cell growth through in vitro experiments. The regulatory relationship between HPVE7 and NCAPH is preliminarily revealed. Methods: 1) 10 fresh tissue specimens of normal cervix and 22 cases of cervical cancer were collected, and the total RNA in the tissue was extracted and reverse transcriptase to cDNA. The total RNA in three cervical cancer cell lines was extracted and reverse transcriptive to cDNA. The expression of NCAPH was detected by real time fluorescence quantitative PCR, and the expression difference in normal cervix and cervical cancer was compared. 65 normal cervix tissues and 149 paraffin specimens of cervical cancer were collected, and the expression of NCAPH protein was detected by immunohistochemical method. Difference in expression of NCAPH protein in cervical and cervical cancer; 2) cultured cervical cancer cell lines HeLa and SiHa, transient transfection to NCAPH siRNA, CCK-8 kits and flat clones to detect the effect of NCAPH on cell proliferation, and the effect of NCAPH on cell migration and invasion by Transwell migration and invasion test; 3) HeLa and HeLa. SiHa, transfection of NCAPH siRNA, using Western Blot and real-time fluorescent quantitative PCR to detect the changes in the expression of related proteins; 4) use JASPAR and PROMO software to predict the transcription factors of the NCAPH promoter region, select the highest transcriptional factor E2F1, and interfere and overexpress the transcription factors in HeLa and SiHa. 5) connect the NCAPH gene promoter sequence to the pGL3-Basic vector, construct the overexpression vector of the transcription factor E2F1, then co transfect the 293T cell, and use the double luciferase reporter detection system to verify whether the transcription factor E2F1 can combine the NCAPH promoter region, activate the NCAPH gene transcription, and 6) to cultivate the RPE1 E7 of the high expression of HPV16E7. The effect of E7 overexpression on the expression of NCAPH and transcription factor E2F1, and the interference of E7 in HeLa and SiHa, to detect the expression of E2F1 in NCAPH and transcription factors in HeLa and SiHa, and to interfere with the expression of the transcription factor E2F1 in RPE1E7 cells. Results: 1) 1) in 3 kinds of cervical cancer Compared with normal cervical tissue, the expression level of NCAPH in cervical cancer tissues was significantly higher than that of normal cervical tissues; 2) CCK-8. The experimental results showed that after interfering with the expression of NCAPH in cervical cancer cells, the proliferation ability of the cells decreased significantly and the clone formation energy decreased significantly; and 3) interfered with the expression of NCAPH in cervical cancer cells. The cell migration and invasion ability were significantly inhibited, the expression level of epithelial related protein ZO-1 increased significantly, while the protein expression level of interrelated leaf related Vimentin, Snail decreased significantly; 4) after interfering with the expression of NCAPH in cervical cancer cells, the total AKT in the cell was not obviously changed, and the expression of AKT protein phosphorylation of serine site was expressed. Significant decrease; 5) transcription factor E2F1 can bind to the promoter region of NCAPH and increase the transcription of NCAPH. The key carcinogenic protein E7 of HPV can regulate the expression of NCAPH downstream by regulating E2F1. Conclusion: NCAPH may participate in the development of cervical cancer as a new oncogene. It can significantly promote the proliferation of cervical cancer cells. Over promotion of EMT enhances the migration and invasion of cervical cancer cells..HPV E7 may activate transcription of NCAPH through transcription factor E2F1 and participate in cervical carcinogenesis.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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