EZH2促进膀胱癌生长机制的研究

发布时间：2018-05-30 04:24

本文选题：EZH2 + miRNA-143　；参考：《南京大学》2014年硕士论文

【摘要】：目的探讨EZH2促进膀胱癌生长的机制。方法以siRNA的方式敲降EZH2；高表达或抑制miRNA-143处理膀胱癌细胞T24和5637,通过细胞生存实验观察膀胱癌细胞的生长情况；克隆形成实验观察肿瘤细胞的增殖能力。以实时定量PCR检测经相应处理的膀胱癌细胞mRNA水平的变化。为了验证miRNA-143是EZH2的靶基因,以染色质免疫共沉淀技术(CHIP)检测EZH2对miR-143的结合情况。提取经敲降EZH2；高表达或抑制miRNA-143处理的T24和5637膀胱癌细胞总蛋白质,应用蛋白质免疫印迹检测法(Western blot)观察细胞增殖蛋白和肿瘤恶性程度蛋白的表达情况。结果体外实验中,以siRNA敲降EZH2,细胞生存实验显示与siCTRL组相比,敲降EZH2后膀胱癌细胞T24和5637的生长受到抑制。克隆形成实验结果显示与siCTRL组相比,敲降EZH2七天后,膀胱癌细胞T24和5637中敲降EZH2细胞克隆团数会下降。Western blot法显示在T24和5637膀胱癌细胞中,与siCTRL组相比,敲降EZH2的效果满意；在敲降EZH2后代表肿瘤恶性程度的蛋白CD44、Notch-1、Sox-2. EZH2靶蛋白H3K27me3的表达量下降,细胞增殖蛋白CyclinD1的表达量亦下降。体外实验中,以siRNA敲降EZH2, Q-PCR显示随着EZH2的敲降,在膀胱癌细胞T24和5637中miRNA-143和miRNA-145的表达量上升。接着我们用CHIP实验来检测EZH2如何作用于miRNA-143,结果显示EZH2通过与miRNA-143启动子区域结合来干扰miRNA-143的表达。提示miRNA-143是EZH2的直接靶基因。体外实验中,以瞬时转染的方式高表达miRNA-143 (mimic miR-143),细胞生存实验显示与对照组相比,高表达miRNA-143后膀胱癌细胞T24和5637的生长受到抑制,克隆形成实验结果显示,在膀胱癌细胞124和5637中高表达miRNA-143细胞克隆团数会逐渐下降。Western blot法显示在T24和5637膀胱癌细胞中,与对照组相比,在高表达miRNA-143后代表肿瘤恶性程度的蛋白CD44、Sox-2的表达量下降,细胞增殖蛋白CyclinD1的表达量亦下降,而EZH2蛋白表达量并未受影响。体外实验中,瞬时转染siRNA敲降EZH2,或瞬时转染抑制miRNA-143,细胞生存实验显示与对照组相比,单独敲降EZH2,会抑制膀胱癌细胞T24和5637的生长；同时敲降EZH2和抑制miRNA-143后膀胱癌细胞T24和5637的生长受到刺激,克隆形成实验结果显示T24细胞siCTRL+CTRL inhibitor组、siEZH2+CTRL inhibitor组和siEZH2+miR-143inhibitor组克隆团数分别为：214.33±3.28、59.33±0.89、134.03±6.11个； 5637细胞siCTRL+CTRL inhibitor组、siEZH2+CTRL inhibitor组和siEZH2+miR-143inhibitor组克隆团数分别为：148.05±2.03、72.00±13.00、105.7±2.40个,提示在膀胱癌细胞T24和5637中抑制miRNA-143可以逆转敲降EZH2引起的细胞克隆团数下降。Western blot法显示在T24和5637膀胱癌细胞中,与siEZH2相比,在抑制miRNA-143后代表肿瘤恶性程度的蛋白CD44、Sox-2的表达量上升,细胞增殖蛋白CyclinDl的表达量亦上升。提示EZH2引起的增殖可以通过改变其下游miRNA-143来逆转。结论EZH2可以通过直接抑制miRNA-143而促进膀胱肿瘤细胞的生长。
[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 卤3.2859.33 卤0.89134.03 卤6.11, and 148.05 卤2.032.00 卤13.00105.7 卤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 卤2.032.00 卤13.00105.7 卤2.40, respectively, and that in siCTRL CTRL inhibitor group was 148.05 卤2.032.00 卤13.00105.7 卤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.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

【共引文献】