调控XIAP的miRNA筛选及其促卵巢癌细胞凋亡的研究

发布时间：2018-11-11 11:42

【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个重要原因是由于患者对抗肿瘤药物出现耐药,即化疗药物所诱导的肿瘤细胞凋亡被抑制。XIAP被认为是迄今为止作用最强的内源性抗凋亡蛋白,在多种肿瘤组织中高表达,但其在卵巢癌组织中表达失调的分子作用机制还不清楚。微小RNA(miRNA)是一类高度保守的非编码小分子RNA,在肿瘤的发生、发展及耐药性的形成中起重要作用。故筛选出与XIAP相互作用的miRNA,阐述其参与凋亡调节的分子机制非常重要,不仅将揭示一个全新的由miRNA介导的遗传信息调控网络,也为肿瘤的早期诊断、疗效评估以及疾病预后的分析提供重要分子依据,为肿瘤治疗提供新的潜在靶点。方法1.免疫组化及免疫印迹检验XIAP在卵巢癌组织及对照卵巢组织中的表达情况;2.在卵巢癌细胞中,用慢病毒过表达XIAP或RNA干扰技术(siRNA)降低XIAP的表达,观察细胞凋亡情况;3.运用生物信息学软件预测靶向XIAP基因3'UTR的miRNA。综合文献报道,从预测的结果中找出在卵巢癌组织中低表达的miRNA,用双荧光素酶报告基因和miRNA表达载体共转染293T细胞方式进行筛选和验证。4.选取经验证的作用效果强的miR-155和miR-137,分别用作用位点突变、荧光定量PCR、免疫印迹、siRNA和细胞凋亡的功能实验进一步明确每个miRNA对顺铂诱导的卵巢癌细胞凋亡的影响及可能机制,并在卵巢癌组织样本及细胞系中对miR-137与XIAP的表达水平进行相关性分析。结果1.XIAP在上皮性卵巢癌组织中高表达。在卵巢癌细胞系SKOV3中过表达XIAP可抑制顺铂诱导的细胞凋亡,而降低XIAP的表达则作用相反。2.综合软件预测与文献报道的结果,发现既与XIAP 3'UTR有相互作用又在卵巢癌组织中低表达的候选miRNA共22个,用双荧光素酶报告基因筛选出其中8个miRNA与XIAP 3'UTR确有相互作用,其中miR-137,miR-155,miR-142及miR-146a的作用明显。3.针对作用很强的miR-155和miR-137,本实验发现它们均可以降低卵巢癌细胞SKOV3、A2780的顺铂IC50值,并促进卵巢癌细胞系及原代培养癌细胞的顺铂所诱导的细胞凋亡。位点突变和凋亡功能回复实验都说明是直接通过作用于XIAP 3'UTR来抑制XIAP蛋白表达的。区别于miR-155,miR-137与XIAP 3'UTR有两个作用位点。进一步分析卵巢癌组织与正常对照样本的结果,显示XIAP在卵巢癌组织中表达水平明显升高,miR-137的表达水平显著降低,并且两者的表达水平负相关。结论XIAP在上皮性卵巢癌组织中表达水平明显升高,并能抑制卵巢癌细胞对顺铂的敏感性,提示XIAP与卵巢癌的发生发展相关,是导致卵巢癌化疗耐药的重要原因之一。低表达的miRNA是导致XIAP在卵巢癌组织中表达升高的重要原因。由于其中的miR-155和miR-137能够促进顺铂所致的细胞凋亡,说明这些miRNA很可能通过调节XIAP的表达,参与了卵巢癌细胞凋亡的调控和卵巢癌化疗耐药的形成,因而这些miRNA的表达异常可能是卵巢癌化疗耐药的机制之一,并可能成为开发新的化疗药物的潜在靶点。
[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.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.31

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