本文選題：EZH2 + miRNA-143��； 參考：《南京大學》2014年碩士論文

【摘要】：目的探討EZH2促進膀胱癌生長的機制。方法以siRNA的方式敲降EZH2；高表達或抑制miRNA-143處理膀胱癌細胞T24和5637,通過細胞生存實驗觀察膀胱癌細胞的生長情況；克隆形成實驗觀察腫瘤細胞的增殖能力。以實時定量PCR檢測經(jīng)相應處理的膀胱癌細胞mRNA水平的變化。為了驗證miRNA-143是EZH2的靶基因,以染色質(zhì)免疫共沉淀技術(CHIP)檢測EZH2對miR-143的結(jié)合情況。提取經(jīng)敲降EZH2；高表達或抑制miRNA-143處理的T24和5637膀胱癌細胞總蛋白質(zhì),應用蛋白質(zhì)免疫印跡檢測法(Western blot)觀察細胞增殖蛋白和腫瘤惡性程度蛋白的表達情況。結(jié)果體外實驗中,以siRNA敲降EZH2,細胞生存實驗顯示與siCTRL組相比,敲降EZH2后膀胱癌細胞T24和5637的生長受到抑制。克隆形成實驗結(jié)果顯示與siCTRL組相比,敲降EZH2七天后,膀胱癌細胞T24和5637中敲降EZH2細胞克隆團數(shù)會下降。Western blot法顯示在T24和5637膀胱癌細胞中,與siCTRL組相比,敲降EZH2的效果滿意；在敲降EZH2后代表腫瘤惡性程度的蛋白CD44、Notch-1、Sox-2. EZH2靶蛋白H3K27me3的表達量下降,細胞增殖蛋白CyclinD1的表達量亦下降。體外實驗中,以siRNA敲降EZH2, Q-PCR顯示隨著EZH2的敲降,在膀胱癌細胞T24和5637中miRNA-143和miRNA-145的表達量上升。接著我們用CHIP實驗來檢測EZH2如何作用于miRNA-143,結(jié)果顯示EZH2通過與miRNA-143啟動子區(qū)域結(jié)合來干擾miRNA-143的表達。提示miRNA-143是EZH2的直接靶基因。體外實驗中,以瞬時轉(zhuǎn)染的方式高表達miRNA-143 (mimic miR-143),細胞生存實驗顯示與對照組相比,高表達miRNA-143后膀胱癌細胞T24和5637的生長受到抑制,克隆形成實驗結(jié)果顯示,在膀胱癌細胞124和5637中高表達miRNA-143細胞克隆團數(shù)會逐漸下降。Western blot法顯示在T24和5637膀胱癌細胞中,與對照組相比,在高表達miRNA-143后代表腫瘤惡性程度的蛋白CD44、Sox-2的表達量下降,細胞增殖蛋白CyclinD1的表達量亦下降,而EZH2蛋白表達量并未受影響。體外實驗中,瞬時轉(zhuǎn)染siRNA敲降EZH2,或瞬時轉(zhuǎn)染抑制miRNA-143,細胞生存實驗顯示與對照組相比,單獨敲降EZH2,會抑制膀胱癌細胞T24和5637的生長；同時敲降EZH2和抑制miRNA-143后膀胱癌細胞T24和5637的生長受到刺激,克隆形成實驗結(jié)果顯示T24細胞siCTRL+CTRL inhibitor組、siEZH2+CTRL inhibitor組和siEZH2+miR-143inhibitor組克隆團數(shù)分別為：214.33±3.28、59.33±0.89、134.03±6.11個； 5637細胞siCTRL+CTRL inhibitor組、siEZH2+CTRL inhibitor組和siEZH2+miR-143inhibitor組克隆團數(shù)分別為：148.05±2.03、72.00±13.00、105.7±2.40個,提示在膀胱癌細胞T24和5637中抑制miRNA-143可以逆轉(zhuǎn)敲降EZH2引起的細胞克隆團數(shù)下降。Western blot法顯示在T24和5637膀胱癌細胞中,與siEZH2相比,在抑制miRNA-143后代表腫瘤惡性程度的蛋白CD44、Sox-2的表達量上升,細胞增殖蛋白CyclinDl的表達量亦上升。提示EZH2引起的增殖可以通過改變其下游miRNA-143來逆轉(zhuǎn)。結(jié)論EZH2可以通過直接抑制miRNA-143而促進膀胱腫瘤細胞的生長。
[Abstract]:Objective to investigate the mechanism of EZH2 promoting bladder cancer growth. Methods siRNA was used to knock down EZH2, high expression or inhibition of miRNA-143 was used to treat bladder cancer cells T24 and 5637, cell survival test was used to observe the growth of bladder cancer cells, and clone formation assay was used to observe the proliferation ability of tumor cells. Real-time quantitative PCR was used to detect the changes of mRNA level in bladder cancer cells. In order to verify that miRNA-143 is the target gene of EZH2, the binding of EZH2 to miR-143 was detected by chromatin immunoprecipitation technique. The total proteins of T24 and 5637 bladder cancer cells treated with miRNA-143 were extracted, and the expression of proliferating protein and malignant degree protein in T24 and 5637 bladder cancer cells were detected by Western blot assay. Results in vitro, siRNA knock down EZH _ 2 and cell survival test showed that the growth of bladder cancer cells T24 and 5637 after knock down EZH2 was inhibited compared with that of siCTRL group. The results of clone formation test showed that compared with siCTRL group, the number of knockout EZH2 clones in bladder cancer cells T24 and 5637 decreased after 7 days of EZH2 knockout. Western blot assay showed that in T24 and 5637 bladder cancer cells, the effect of EZH2 knockout was satisfactory compared with siCTRL group. After knocking down EZH2, the protein CD _ 4 / Notch-1 / Sox-2 represents the malignancy of the tumor. The expression of EZH2 target protein H3K27me3 and cell proliferating protein CyclinD1 decreased. In vitro, the expression of miRNA-143 and miRNA-145 in bladder cancer cell line T24 and 5637 was increased by siRNA knockdown and Q-PCR. Then we use CHIP to detect how EZH2 acts on miRNA-143. The results show that EZH2 interferes with the expression of miRNA-143 by binding to miRNA-143 promoter region. The results suggest that miRNA-143 is a direct target gene of EZH2. In vitro, miRNA-143 mimic miR-143 was overexpressed by transient transfection. Cell survival assay showed that the growth of bladder cancer cells T24 and 5637 was inhibited after overexpression of miRNA-143. The number of high expression miRNA-143 clones in bladder cancer cells 124 and 5637 decreased gradually. Western blot assay showed that in T24 and 5637 bladder cancer cells, compared with the control group, the expression of CD44mSox-2, a protein representing the malignancy of tumor, decreased after high expression of miRNA-143. The expression of proliferating protein CyclinD1 was also decreased, but the expression of EZH2 protein was not affected. In vitro, transient transfection of siRNA knocked down EZH2, or transient transfection inhibited miRNA-143. Compared with the control group, knockdown alone inhibited the growth of bladder cancer cells T24 and 5637. The growth of bladder cancer cells T24 and 5637 was stimulated by knocking down EZH2 and inhibiting the growth of T24 and 5637 cells after miRNA-143. The results of clone formation test showed that the number of clone groups in siCTRL CTRL inhibitor group and siEZH2 miR-143inhibitor group were respectively: 1: 214.33 鹵3.2859.33 鹵0.89134.03 鹵6.11, and 148.05 鹵2.032.00 鹵13.00105.7 鹵2.40 in siCTRL CTRL inhibitor group and siEZH2 miR-143inhibitor group, respectively, in T24 cell group, the number of clones in T24 cell group was 148.05 鹵2.032.00 鹵13.00105.7 鹵2.40, respectively, and that in siCTRL CTRL inhibitor group was 148.05 鹵2.032.00 鹵13.00105.7 鹵2.40, respectively. It was suggested that inhibition of miRNA-143 in T24 and 5637 cells could reverse the decrease in the number of cell clones induced by knock down EZH2. Western blot assay showed that in T24 and 5637 bladder cancer cells, compared with siEZH2, inhibition of miRNA-143 could reverse the decrease of cell clone number induced by knock down EZH2. After inhibition of miRNA-143, the expression of CD4N Sox-2, which represents the malignancy of tumor, was increased, and the expression of cell proliferating protein CyclinDl was also increased. The results suggest that the proliferation induced by EZH2 can be reversed by changing its downstream miRNA-143. Conclusion EZH2 can promote the growth of bladder tumor cells by directly inhibiting miRNA-143.
【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.14

【共引文獻】

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