HPV相关miR-3156-3p的表达在宫颈癌中作用机制的研究

发布时间：2018-04-03 09:51

本文选题：微小RNA　切入点：宫颈癌　出处：《青岛大学》2017年博士论文

【摘要】：研究目的:宫颈癌的发生率在女性常见恶性肿瘤中列第三位,尤其在发展中国家高发,严重威胁女性健康。已知宫颈癌与高危型人乳头瘤病毒(HR-HPV),尤其HPV16/18型的持续感染有关,而HR-HPV癌蛋白E6、E7通过不同的作用机制在宫颈癌的发生、发展中扮演重要角色。微小RNA(microRNA,miRNA)是长约20个核苷酸的非编码小RNA,可调控大量的目的基因mRNA,发挥生物学功能。研究证实,miRNA与多种肿瘤的发病机制有关,包括宫颈癌。近年来一些研究发现,人乳头瘤病毒(HPV)可以通过E6、E7癌蛋白影响细胞中miRNA的表达。因此,本研究的目的是探讨HPV感染与miRNA表达异常的关系,及其在宫颈癌发生、发展中的作用及作用机制。研究方法:1.构建HPV16E6、E7和E6/E7基因稳定转染的HPV阴性HT-3和C33A宫颈癌细胞系,采用miRNA表达谱芯片检测和筛选HPV16 E6/E7基因稳定转染前后差异表达的miRNA,采用qRT-PCR方法验证芯片筛选结果。2.选取HPV16 E6/E7转染后显著差异表达的miR-3156-3p作为候选miRNA,qRT-PCR方法分别检测miR-3156-3p在HPV阳性、阴性宫颈癌组织及正常宫颈上皮组织内的表达水平。3.在宫颈癌细胞系中瞬时转染miR-3156-3p的模拟物、抑制剂及相应阴性对照,以过表达或降调CaSki、SiHa和HeLa细胞中miR-3156-3p水平,利用CCK8细胞增殖实验、流式细胞凋亡实验、transwell小室迁移侵袭实验、matrigel血管形成实验在细胞水平进行功能实验。4.生物信息软件预测miR-3156-3p潜在的靶基因为SLC6A6,qRT-PCR和Western-blot方法分别检测miR-3156-3p过表达或降调的CaSki细胞和SiHa细胞中SLC6A6 mRNA和蛋白的表达情况,进一步采用双荧光素酶报告实验验证SLC6A6 mRNA3'非翻译区(3'UTR)是否存在miR-3156-3p的结合位点。5.qRT-PCR和免疫组织化学实验分别检测SLC6A6在宫颈癌组织和对照正常宫颈组织中mRNA和蛋白的表达水平。采用亚硫酸氢盐-基因组测序法(Bisulfite Genomic Sequencing,BGS)联合TA克隆测序检测HPV阳性、HPV阴性宫颈癌细胞系及正常宫颈组织与宫颈癌组织中SLC6A6基因启动子区的甲基化状态。结果:1.miRNA表达谱芯片筛选出6个差异表达miRNA(miR-3156-3p、miR-4779-3p、miR-4779-3p、miR-6841-3p、miR-454-5p和miR-656-5p),在HPV16E6/E7基因稳定转染的HT-3细胞中均较空载对照组表达降低。细胞系qRT-PCR实验结果证实miR-3156-3p在HPV16 E6、E7基因转染的HT-3和C33A细胞中较空载对照组表达降低,与芯片筛选结果一致。2.组织学qRT-PCR结果发现,miR-3156-3p在宫颈癌组织中表达较正常宫颈组织降低,且HPV16/18阳性宫颈癌组织较HPV阴性癌组织中miR-3156-3p表达显著降低。3.体外细胞功能实验验证,miR-3156-3p具有抑制宫颈癌细胞增殖、促进凋亡,抑制肿瘤细胞迁移侵袭及血管形成的能力。4.Western-blot结果提示,蛋白水平miR-3156-3p可降调宫颈癌细胞中SLC6A6蛋白表达,但qRT-PCR结果显示,在mRNA水平miR-3156-3p的表达对SLC6A6mRNA无影响。双荧光素酶报告实验提示miR-3156-3p模拟物对SLC6A6野生型载体的报告荧光较对照组明显下调,而SLC6A6突变型载体中的报告荧光无明显变化。5.宫颈癌组织中SLC6A6 mRNA表达水平较正常宫颈组织升高,免疫组织化学染色检测发现宫颈癌组织中SLC6A6蛋白的表达水平在宫颈癌组织中明显高于正常宫颈组织。亚硫酸盐基因组测序法(BGS)结果提示SLC6A6启动子在HPV阳性与阴性宫颈癌细胞系、宫颈癌组织及正常宫颈上皮组织均呈低甲基化状态。结论:1.miR-3156-3p在宫颈癌中表达降低,可能与HR-HPV感染相关;2.miR-3156-3p可促进宫颈癌细胞凋亡,抑制细胞增殖、迁移、侵袭以及血管形成能力;3.miR-3156-3p可能通过从转录后水平负调控其靶基因SLC6A6蛋白表达,在宫颈癌中发挥抑癌作用。
[Abstract]:Objective: To study the incidence of malignant tumors in women ranked third in cervical cancer, especially in developing countries, a serious threat to women's health. The known cervical cancer and high-risk human papilloma virus (HR-HPV), especially the persistent infection of HPV16/18 type, and HR-HPV cancer protein E6, E7 through different mechanisms in the incidence of cervical cancer, play an important role in the development of micro RNA. (microRNA, miRNA) is a small non encoding RNA of about 20 nucleotides in length, which can regulate target gene of mRNA, its biological function. Studies confirmed that the pathogenesis of miRNA and a variety of tumors, including cervical cancer. Some studies have found in recent years. And the human papilloma virus (HPV) by E6, the expression of E7 protein in miRNA cell carcinoma effect. Therefore, the purpose of this study is to investigate the infection of HPV and the abnormal expression of miRNA and its relationship in cervical cancer and its role in the development and The mechanism. Methods: 1. construction of HPV16E6, HT-3 and C33A HPV negative cervical cancer cell lines E7 and E6/E7 gene transfection, using miRNA expression before and after the detection and screening of HPV16 transfected E6/E7 gene microarray miRNA, using the method of qRT-PCR expression significantly validated the microarray results of selected.2. HPV16 E6/E7 transfected miR-3156-3p as a candidate of miRNA, qRT-PCR were detected in miR-3156-3p positive HPV, mimics the expression level of.3. negative cervical carcinoma and normal cervical epithelial tissue in transient in cervical carcinoma cells transfected with miR-3156-3p inhibitor, and the corresponding negative control, the overexpression or depletion of CaSki, SiHa and miR-3156-3p levels in HeLa cells by CCK8 experiment cell proliferation and apoptosis by flow cytometry experiments, Transwell cell migration and invasion experiment, Matrigel experimental angiogenesis at the cellular level in real function Check the.4. bioinformatics software predicted target genes of miR-3156-3p potential because of SLC6A6, qRT-PCR and Western-blot were used to detect the expression of SLC6A6 mRNA and protein in CaSki cells and SiHa cells overexpressing miR-3156-3p or down-regulation in the further experiment using dual luciferase SLC6A6 mRNA3'untranslated region (3'UTR) the presence of miR-3156-3p binding sites and.5.qRT-PCR immunohistochemistry was used to detect the expression level of SLC6A6 mRNA in cervical cancer tissue and normal cervical tissue and protein. Using bisulfite genomic sequencing (Bisulfite Genomic, Sequencing, BGS) combined with TA sequencing to detect HPV positive and SLC6A6 HPV negative cervical cancer cell lines and normal cervical tissue and cervical cancer tissue gene promoter methylation status. Results: 1.miRNA microarray screened 6 differentially expressed miRNA (miR-3156-3p, M IR-4779-3p, miR-4779-3p, miR-6841-3p, miR-454-5p and miR-656-5p), the HPV16E6/E7 gene stable transfected HT-3 cells were lower than control group. The decreased expression of load cell line qRT-PCR miR-3156-3p in the HPV16 experimental results show that E6, E7 gene transfected HT-3 and C33A cells compared with the control group decreased expression of empty load, consistent.2. histological qRT-PCR results with the chip screening, the expression of miR-3156-3p in cervical cancer compared with normal cervical tissue decreased, and HPV16/18 positive cervical cancer with HPV negative cancer tissue miR-3156-3p expression significantly decreased.3. cell function in vitro experiments, miR-3156-3p could inhibit the proliferation of cervical cancer cell apoptosis, inhibit tumor cell invasion and angiogenesis ability of.4.Western-blot results suggest that the protein level of miR-3156-3p expression of SLC6A6 protein in cervical cancer cells. However, qRT-PCR results showed that, in mRNA The expression level of miR-3156-3p had no effect on SLC6A6mRNA. Dual luciferase reporter experiments suggest that the analogue of miR-3156-3p report fluorescence of SLC6A6 wild type carrier was significantly reduced compared with the control group, and the report of fluorescent SLC6A6 mutant in the carrier level is no normal cervical tissue increased SLC6A6 mRNA expression changes of.5. in cervical carcinoma, immunohistochemistry staining the expression level of SLC6A6 protein in cervical carcinoma was significantly higher than that in normal cervical tissue in cervical cancer. Bisulfite genomic sequencing (BGS) results indicated that SLC6A6 promoter in HPV positive and negative cervical cancer cell line, cervical carcinoma and normal cervical epithelial tissue showed low methylation status. Conclusion: the decreased expression of 1.miR-3156-3p in cervical cancer, may be associated with HR-HPV infection; 2.miR-3156-3p can promote the apoptosis of cervical cancer cells, inhibition of cell proliferation, migration, invasion As well as angiogenesis, 3.miR-3156-3p may play a role in inhibiting cancer in cervical cancer by negatively regulating the expression of its target gene SLC6A6 protein from post transcriptional level.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33

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