本文選題：微小RNA　切入點：宮頸癌　出處：《青島大學(xué)》2017年博士論文

【摘要】：研究目的:宮頸癌的發(fā)生率在女性常見惡性腫瘤中列第三位,尤其在發(fā)展中國家高發(fā),嚴重威脅女性健康。已知宮頸癌與高危型人乳頭瘤病毒(HR-HPV),尤其HPV16/18型的持續(xù)感染有關(guān),而HR-HPV癌蛋白E6、E7通過不同的作用機制在宮頸癌的發(fā)生、發(fā)展中扮演重要角色。微小RNA(microRNA,miRNA)是長約20個核苷酸的非編碼小RNA,可調(diào)控大量的目的基因mRNA,發(fā)揮生物學(xué)功能。研究證實,miRNA與多種腫瘤的發(fā)病機制有關(guān),包括宮頸癌。近年來一些研究發(fā)現(xiàn),人乳頭瘤病毒(HPV)可以通過E6、E7癌蛋白影響細胞中miRNA的表達。因此,本研究的目的是探討HPV感染與miRNA表達異常的關(guān)系,及其在宮頸癌發(fā)生、發(fā)展中的作用及作用機制。研究方法:1.構(gòu)建HPV16E6、E7和E6/E7基因穩(wěn)定轉(zhuǎn)染的HPV陰性HT-3和C33A宮頸癌細胞系,采用miRNA表達譜芯片檢測和篩選HPV16 E6/E7基因穩(wěn)定轉(zhuǎn)染前后差異表達的miRNA,采用qRT-PCR方法驗證芯片篩選結(jié)果。2.選取HPV16 E6/E7轉(zhuǎn)染后顯著差異表達的miR-3156-3p作為候選miRNA,qRT-PCR方法分別檢測miR-3156-3p在HPV陽性、陰性宮頸癌組織及正常宮頸上皮組織內(nèi)的表達水平。3.在宮頸癌細胞系中瞬時轉(zhuǎn)染miR-3156-3p的模擬物、抑制劑及相應(yīng)陰性對照,以過表達或降調(diào)CaSki、SiHa和HeLa細胞中miR-3156-3p水平,利用CCK8細胞增殖實驗、流式細胞凋亡實驗、transwell小室遷移侵襲實驗、matrigel血管形成實驗在細胞水平進行功能實驗。4.生物信息軟件預(yù)測miR-3156-3p潛在的靶基因為SLC6A6,qRT-PCR和Western-blot方法分別檢測miR-3156-3p過表達或降調(diào)的CaSki細胞和SiHa細胞中SLC6A6 mRNA和蛋白的表達情況,進一步采用雙熒光素酶報告實驗驗證SLC6A6 mRNA3'非翻譯區(qū)(3'UTR)是否存在miR-3156-3p的結(jié)合位點。5.qRT-PCR和免疫組織化學(xué)實驗分別檢測SLC6A6在宮頸癌組織和對照正常宮頸組織中mRNA和蛋白的表達水平。采用亞硫酸氫鹽-基因組測序法(Bisulfite Genomic Sequencing,BGS)聯(lián)合TA克隆測序檢測HPV陽性、HPV陰性宮頸癌細胞系及正常宮頸組織與宮頸癌組織中SLC6A6基因啟動子區(qū)的甲基化狀態(tài)。結(jié)果:1.miRNA表達譜芯片篩選出6個差異表達miRNA(miR-3156-3p、miR-4779-3p、miR-4779-3p、miR-6841-3p、miR-454-5p和miR-656-5p),在HPV16E6/E7基因穩(wěn)定轉(zhuǎn)染的HT-3細胞中均較空載對照組表達降低。細胞系qRT-PCR實驗結(jié)果證實miR-3156-3p在HPV16 E6、E7基因轉(zhuǎn)染的HT-3和C33A細胞中較空載對照組表達降低,與芯片篩選結(jié)果一致。2.組織學(xué)qRT-PCR結(jié)果發(fā)現(xiàn),miR-3156-3p在宮頸癌組織中表達較正常宮頸組織降低,且HPV16/18陽性宮頸癌組織較HPV陰性癌組織中miR-3156-3p表達顯著降低。3.體外細胞功能實驗驗證,miR-3156-3p具有抑制宮頸癌細胞增殖、促進凋亡,抑制腫瘤細胞遷移侵襲及血管形成的能力。4.Western-blot結(jié)果提示,蛋白水平miR-3156-3p可降調(diào)宮頸癌細胞中SLC6A6蛋白表達,但qRT-PCR結(jié)果顯示,在mRNA水平miR-3156-3p的表達對SLC6A6mRNA無影響。雙熒光素酶報告實驗提示miR-3156-3p模擬物對SLC6A6野生型載體的報告熒光較對照組明顯下調(diào),而SLC6A6突變型載體中的報告熒光無明顯變化。5.宮頸癌組織中SLC6A6 mRNA表達水平較正常宮頸組織升高,免疫組織化學(xué)染色檢測發(fā)現(xiàn)宮頸癌組織中SLC6A6蛋白的表達水平在宮頸癌組織中明顯高于正常宮頸組織。亞硫酸鹽基因組測序法(BGS)結(jié)果提示SLC6A6啟動子在HPV陽性與陰性宮頸癌細胞系、宮頸癌組織及正常宮頸上皮組織均呈低甲基化狀態(tài)。結(jié)論:1.miR-3156-3p在宮頸癌中表達降低,可能與HR-HPV感染相關(guān);2.miR-3156-3p可促進宮頸癌細胞凋亡,抑制細胞增殖、遷移、侵襲以及血管形成能力;3.miR-3156-3p可能通過從轉(zhuǎn)錄后水平負調(diào)控其靶基因SLC6A6蛋白表達,在宮頸癌中發(fā)揮抑癌作用。
[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.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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