MiR-506调控EZH2-β-catenin信号通路对浆液性卵巢癌药物敏感性的影响

发布时间：2018-07-13 17:30

【摘要】：目的:化疗耐药的产生是肿瘤治疗中面对的主要挑战。上皮性卵巢癌死亡率高、预后差,患者多死于肿瘤复发和耐药。肿瘤耐药与多种因素相关,mi RNA可能与耐药密切相关。我们发现mi R-506是一种与化疗耐药相关的上皮-间质转化的有效抑制剂。我们认为mi R-506影响了高级别浆液性卵巢癌的化疗反应。本研究中,我们讨论mi R-506是否能增加浆液性卵巢癌细胞顺铂和PARP抑制剂的敏感性及通过检测mi R-506对靶基因EZH2-β-catenin信号通路的调控作用,阐述mi R-506发挥药物增敏作用的机制。方法:本研究分为三个部分进行:1.通过荧光素酶报告系统证明mi R-506通过直接抑制EZH2的3’非编码区(3’-untranslated region 3’-UTR),从而下调β-catenin的表达。在卵巢癌细胞系Hey A8过表达mi R-506,western blot检测EZH2和β-catenin蛋白表达。检测mi R-506是否能够下调EZH2和β-catenin水平。2.转染不含3’UTR的EZH2和mi R-506或对照mi R mimics,以及应用si RNA敲除EZH2,观察EZH2和β-catenin蛋白水平及顺铂和PARP抑制剂药物敏感性的变化。3.分别过表达β-catenin活性载体和应用β-catenin抑制剂FH535处理卵巢癌细胞后,观察药物敏感性的变化以及过表达β-catenin对mi R-506调控药物敏感性的影响。结果:1.在卵巢癌细胞系Hey A8中与对照组相比mi R-506可显著下调EZH2和β-catenin的蛋白水平;搜索Target Scan(http://www.targetscan.org/)得到了mi R-506与EZH2的结合位点;荧光素酶报告实验中与阴性对照相比,共转染mi R-506mimcs和EZH2质粒后发现,在过表达mi R-506时,野生型3’-UTR报告载体的荧光与突变组的荧光相比明显减弱。(P0.01)。2.卵巢癌Hey A8/SKOV3细胞系与对照组相比,下调EZH2后,β-catenin蛋白水平显著下降,加入顺铂/奥拉帕尼后在450nm处的OD值明显下降;上调EZH2后,β-catenin蛋白水平显著升高,加入顺铂/奥拉帕尼后在450nm处的OD值明显升高;过表达不含3'-UTR的EZH2可以部分补救mi R-506对顺铂和奥拉帕尼敏感性的影响。(P0.05)。3.卵巢癌Hey A8/SKOV3细胞系与对照组相比,下调β-catenin后,加入顺铂/奥拉帕尼在450nm处的OD值明显下降,细胞的克隆形成率明显降低;上调β-catenin后,加入顺铂/奥拉帕尼在450nm处的OD值明显升高,细胞的克隆形成率明显增加;过表达不含3'-UTR的β-catenin可以部分补救mi R-506对顺铂和奥拉帕尼敏感性的影响。(P0.05)。结论:通过本实验,可以得出以下几个结论:1.EZH2是mi R-506的直接靶点。2.Mi R-506可以抑制EZH2-β-catenin信号通路。3.Mi R-506发挥药物增敏作用的机制是通过靶向EZH2抑制β-catenin信号通路。
[Abstract]:Objective: the production of chemotherapeutic resistance is a major challenge in tumor treatment. Epithelial ovarian cancer has a high mortality and poor prognosis. Most of the patients die from tumor recurrence and drug resistance. Tumor resistance may be associated with multiple factors, such as MMI RNA and drug resistance. We found that mi-506 is an effective inhibitor of epithelial-interstitial transformation associated with chemotherapeutic resistance. We believe that mi R 506 affects the chemotherapeutic response of high grade serous ovarian cancer. In this study, we discussed whether mi R-506 could increase the sensitivity of cisplatin and PARP inhibitors in serous ovarian cancer cells, and examine the role of mi R-506 in the regulation of target gene EZH2- 尾 -catenin signaling pathway, and elucidate the mechanism of the drug sensitizing effect of mi R-506. Methods: this study was divided into three parts: 1. It was proved by luciferase reporting system that mi R-506 down-regulated the expression of 尾 -catenin by directly inhibiting the 3'non-coding region (3'-untranslated region 3'-UTR) of EZH2. The expression of EZH2 and 尾 -catenin was detected in ovarian cancer cell line Hei A8 overexpression mi R-506western blot. Whether or not mi R-506 can down-regulate EZH2 and 尾 -catenin levels. Transfection of EZH2 and mi R-506 or control mi R mimicswithout 3-UTR, and use of si RNA knockout of EZH2 to observe the changes of EZH2 and 尾 -catenin protein levels and drug sensitivity of cisplatin and PARP inhibitors. After overexpression of 尾 -catenin and treatment of ovarian cancer cells with 尾 -catenin inhibitor FH535, the changes of drug sensitivity and the effect of overexpression of 尾 -catenin on the drug sensitivity of mi R-506 were observed. The result is 1: 1. The protein levels of EZH2 and 尾 -catenin were significantly down-regulated by mi R-506 in ovarian cancer cell line Hey A8, the binding sites of mi R-506 to EZH2 were obtained by searching Target scan (http://www.targetscan.org/), and the luciferase report was compared with negative control. After co-transfection of miR-506 mimcs and EZH2 plasmids, it was found that the fluorescence of wild-type 3N-UTR reporter vector was significantly weaker than that of mutant group. (P0.01) .2. Compared with the control group, the 尾 -catenin protein level decreased significantly after down-regulating EZH2, and the OD value of 尾 -catenin at 450nm was significantly decreased after adding cisplatin / olapanil, and 尾 -catenin protein level increased significantly after upregulation of EZH2. After addition of cisplatin / olapanil, the OD value at 450nm was significantly increased, and the overexpression of EZH2 without 3H-UTR could partly remedy the effect of mi R-506 on the sensitivity of cisplatin and olapanil. (P0.05) .3. After down-regulating 尾 -catenin, the OD value of Cisplatin / olapanil at 450nm was significantly decreased and the cell clone formation rate was significantly decreased after up-regulation of 尾 -catenin in ovarian cancer cell line Hey A _ 8 / SKOV3 compared with control group. The OD value of cisplatin / olapanil at 450nm was significantly increased, and the colony formation rate was significantly increased, and 尾 -catenin, which did not contain 3H-UTR, could partly remedy the effect of miR-506 on the sensitivity of cisplatin and olapanil (P0.05). Conclusion: through this experiment, we can draw the following conclusions: 1. EZH2 is the direct target of mi R-506. 2. Mi R-506 can inhibit EZH2- 尾 -catenin signaling pathway. 3. Mi R-506 inhibits 尾 -catenin signaling pathway by targeting EZH2.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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