【摘要】：目的:化療耐藥的產生是腫瘤治療中面對的主要挑戰(zhàn)。上皮性卵巢癌死亡率高、預后差,患者多死于腫瘤復發(fā)和耐藥。腫瘤耐藥與多種因素相關,mi RNA可能與耐藥密切相關。我們發(fā)現mi R-506是一種與化療耐藥相關的上皮-間質轉化的有效抑制劑。我們認為mi R-506影響了高級別漿液性卵巢癌的化療反應。本研究中,我們討論mi R-506是否能增加漿液性卵巢癌細胞順鉑和PARP抑制劑的敏感性及通過檢測mi R-506對靶基因EZH2-β-catenin信號通路的調控作用,闡述mi R-506發(fā)揮藥物增敏作用的機制。方法:本研究分為三個部分進行:1.通過熒光素酶報告系統證明mi R-506通過直接抑制EZH2的3’非編碼區(qū)(3’-untranslated region 3’-UTR),從而下調β-catenin的表達。在卵巢癌細胞系Hey A8過表達mi R-506,western blot檢測EZH2和β-catenin蛋白表達。檢測mi R-506是否能夠下調EZH2和β-catenin水平。2.轉染不含3’UTR的EZH2和mi R-506或對照mi R mimics,以及應用si RNA敲除EZH2,觀察EZH2和β-catenin蛋白水平及順鉑和PARP抑制劑藥物敏感性的變化。3.分別過表達β-catenin活性載體和應用β-catenin抑制劑FH535處理卵巢癌細胞后,觀察藥物敏感性的變化以及過表達β-catenin對mi R-506調控藥物敏感性的影響。結果:1.在卵巢癌細胞系Hey A8中與對照組相比mi R-506可顯著下調EZH2和β-catenin的蛋白水平;搜索Target Scan(http://www.targetscan.org/)得到了mi R-506與EZH2的結合位點;熒光素酶報告實驗中與陰性對照相比,共轉染mi R-506mimcs和EZH2質粒后發(fā)現,在過表達mi R-506時,野生型3’-UTR報告載體的熒光與突變組的熒光相比明顯減弱。(P0.01)。2.卵巢癌Hey A8/SKOV3細胞系與對照組相比,下調EZH2后,β-catenin蛋白水平顯著下降,加入順鉑/奧拉帕尼后在450nm處的OD值明顯下降;上調EZH2后,β-catenin蛋白水平顯著升高,加入順鉑/奧拉帕尼后在450nm處的OD值明顯升高;過表達不含3'-UTR的EZH2可以部分補救mi R-506對順鉑和奧拉帕尼敏感性的影響。(P0.05)。3.卵巢癌Hey A8/SKOV3細胞系與對照組相比,下調β-catenin后,加入順鉑/奧拉帕尼在450nm處的OD值明顯下降,細胞的克隆形成率明顯降低;上調β-catenin后,加入順鉑/奧拉帕尼在450nm處的OD值明顯升高,細胞的克隆形成率明顯增加;過表達不含3'-UTR的β-catenin可以部分補救mi R-506對順鉑和奧拉帕尼敏感性的影響。(P0.05)。結論:通過本實驗,可以得出以下幾個結論:1.EZH2是mi R-506的直接靶點。2.Mi R-506可以抑制EZH2-β-catenin信號通路。3.Mi R-506發(fā)揮藥物增敏作用的機制是通過靶向EZH2抑制β-catenin信號通路。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.31

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