【摘要】：研究背景及目的上皮性卵巢癌患者五年生存率低的一個重要原因是由于患者對抗腫瘤藥物出現耐藥,即化療藥物所誘導的腫瘤細胞凋亡被抑制。XIAP被認為是迄今為止作用最強的內源性抗凋亡蛋白,在多種腫瘤組織中高表達,但其在卵巢癌組織中表達失調的分子作用機制還不清楚。微小RNA(miRNA)是一類高度保守的非編碼小分子RNA,在腫瘤的發(fā)生、發(fā)展及耐藥性的形成中起重要作用。故篩選出與XIAP相互作用的miRNA,闡述其參與凋亡調節(jié)的分子機制非常重要,不僅將揭示一個全新的由miRNA介導的遺傳信息調控網絡,也為腫瘤的早期診斷、療效評估以及疾病預后的分析提供重要分子依據,為腫瘤治療提供新的潛在靶點。方法1.免疫組化及免疫印跡檢驗XIAP在卵巢癌組織及對照卵巢組織中的表達情況;2.在卵巢癌細胞中,用慢病毒過表達XIAP或RNA干擾技術(siRNA)降低XIAP的表達,觀察細胞凋亡情況;3.運用生物信息學軟件預測靶向XIAP基因3'UTR的miRNA。綜合文獻報道,從預測的結果中找出在卵巢癌組織中低表達的miRNA,用雙熒光素酶報告基因和miRNA表達載體共轉染293T細胞方式進行篩選和驗證。4.選取經驗證的作用效果強的miR-155和miR-137,分別用作用位點突變、熒光定量PCR、免疫印跡、siRNA和細胞凋亡的功能實驗進一步明確每個miRNA對順鉑誘導的卵巢癌細胞凋亡的影響及可能機制,并在卵巢癌組織樣本及細胞系中對miR-137與XIAP的表達水平進行相關性分析。結果1.XIAP在上皮性卵巢癌組織中高表達。在卵巢癌細胞系SKOV3中過表達XIAP可抑制順鉑誘導的細胞凋亡,而降低XIAP的表達則作用相反。2.綜合軟件預測與文獻報道的結果,發(fā)現既與XIAP 3'UTR有相互作用又在卵巢癌組織中低表達的候選miRNA共22個,用雙熒光素酶報告基因篩選出其中8個miRNA與XIAP 3'UTR確有相互作用,其中miR-137,miR-155,miR-142及miR-146a的作用明顯。3.針對作用很強的miR-155和miR-137,本實驗發(fā)現它們均可以降低卵巢癌細胞SKOV3、A2780的順鉑IC50值,并促進卵巢癌細胞系及原代培養(yǎng)癌細胞的順鉑所誘導的細胞凋亡。位點突變和凋亡功能回復實驗都說明是直接通過作用于XIAP 3'UTR來抑制XIAP蛋白表達的。區(qū)別于miR-155,miR-137與XIAP 3'UTR有兩個作用位點。進一步分析卵巢癌組織與正常對照樣本的結果,顯示XIAP在卵巢癌組織中表達水平明顯升高,miR-137的表達水平顯著降低,并且兩者的表達水平負相關。結論XIAP在上皮性卵巢癌組織中表達水平明顯升高,并能抑制卵巢癌細胞對順鉑的敏感性,提示XIAP與卵巢癌的發(fā)生發(fā)展相關,是導致卵巢癌化療耐藥的重要原因之一。低表達的miRNA是導致XIAP在卵巢癌組織中表達升高的重要原因。由于其中的miR-155和miR-137能夠促進順鉑所致的細胞凋亡,說明這些miRNA很可能通過調節(jié)XIAP的表達,參與了卵巢癌細胞凋亡的調控和卵巢癌化療耐藥的形成,因而這些miRNA的表達異�？赡苁锹殉舶┗熌退幍臋C制之一,并可能成為開發(fā)新的化療藥物的潛在靶點。
[Abstract]:One important reason for the low five-year survival rate of the study background and target epithelial ovarian cancer is that the patient's resistance to the tumor drug, i.e., the tumor cell apoptosis induced by the chemotherapy drug, is suppressed. XIAP is considered to be the most potent endogenous anti-apoptotic protein to date and is highly expressed in a variety of tumor tissues, but the molecular mechanism of the expression of disorders in ovarian cancer tissues is not clear. MicroRNA (miRNA) is a highly conserved non-coding small-molecule RNA, which plays an important role in the formation, development and drug resistance of the tumor. Therefore, the miRNAs interacting with the XIAP are screened, and the molecular mechanism involved in the regulation of apoptosis is very important, not only a novel miRNA-mediated genetic information regulation network is disclosed, but also an important molecular basis is provided for the early diagnosis of the tumor, the evaluation of the curative effect and the analysis of the prognosis of the disease, and provides a new potential target for tumor therapy. Method 1. The expression of XIAP in ovarian and control ovarian tissues was examined by immunohistochemistry and immunoblotting. In ovarian cancer cells, the expression of XIAP was reduced with lentiviral overexpression of XIAP or RNA interference technology (siRNA), and the cell apoptosis was observed; 3. The miRNAs targeting the XIAP gene 3 'UTR were predicted using bioinformatics software. According to the comprehensive literature, the low-expression miRNAs in the ovarian cancer tissues were identified from the predicted results, and 293T cells were co-transfected with the double-luciferase reporter gene and the miRNA expression vector for screening and verification. selecting the miR-155 and the miR-137 with strong action effect, respectively using the functional experiments of the action site mutation, the fluorescence quantitative PCR, the immunoblotting, the siRNA and the cell apoptosis to further clarify the effect of each miRNA on the apoptosis of the cisplatin-induced ovarian cancer cell and the possible mechanism, The expression levels of miR-137 and XIAP in ovarian cancer tissue samples and cell lines were analyzed. Results 1. XIAP was highly expressed in epithelial ovarian cancer tissues. Overexpression of XIAP in ovarian cancer cell line SKOV3 can inhibit the apoptosis of cisplatin-induced cells, while reducing the expression of XIAP is opposite. The results of the comprehensive software prediction and the literature report show that there are 22 candidate miRNAs that interact with the XIAP 3' UTR and low expression in the ovarian cancer tissues, and 8 miRNAs are screened by the double-luciferase reporter gene, and the action of the miR-137, the miR-155, the miR-142 and the miR-146a is obvious. In response to the very strong miR-155 and miR-137, the experiments have found that they can reduce the cisplatin IC50 value of the ovarian cancer cell SKOV3 and A2780, and promote the cell apoptosis induced by the cisplatin of the ovarian cancer cell line and the primary culture cancer cell. Both site mutation and apoptotic function response experiments demonstrated the direct inhibition of XIAP protein expression by acting on XIAP 3 'UTR. The difference between miR-155, miR-137 and XIAP 3' UTR has two active sites. The results showed that the expression level of XIAP was significantly higher in the ovarian cancer tissues, and the expression level of miR-137 was significantly reduced, and the expression level of the two was negatively correlated. Conclusion The expression of XIAP in epithelial ovarian cancer is significantly higher, and it can inhibit the sensitivity of the ovarian cancer cell to cisplatin, and it is suggested that XIAP is related to the development of ovarian cancer, which is one of the most important reasons leading to the chemotherapy resistance of ovarian cancer. Low-expression miRNAs are an important cause of the increase in the expression of XIAP in ovarian cancer tissues. Because of the fact that the miR-155 and the miR-137 are capable of promoting the apoptosis of the cells caused by cisplatin, the miRNA is very likely to be involved in the regulation of the apoptosis of the ovarian cancer cells and the formation of the ovarian cancer chemotherapy resistance by adjusting the expression of the XIAP, so that the expression of the miRNAs can be one of the mechanisms of the chemotherapy resistance of the ovarian cancer, and may be a potential target for developing new chemotherapeutic agents.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R737.31

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