人工合成小分子双链RNA通过激活p21基因的表达对膀胱癌细胞的抑制作用

发布时间：2018-05-24 16:37

本文选题：miR-1236-3p + miR-370-5p　；参考：《华中科技大学》2016年硕士论文

【摘要】：第一部分外源性和内源性小分子dsRNA对膀胱癌细胞中p21蛋白激活表达能力的比较目的:人工合成分别与miR-1236-3p、miR-370-5p所作用的p21基因启动子序列区域完全互补配对的8对小分子dsRNA:dsP21-242、dsP21-243、dsP21-244、dsP21-245和dsP21-552、dsP21-553、dsP21-554、dsP21-555,分别观察其与miR-1236-3p、miR-370-5p在上调人膀胱癌细胞系(T24和EJ)中抑癌基因p21~(WAF1)/CIP1表达能力的不同。方法:8对dsRNA通过参照sa RNA的设计原则设计合成。分别转染dsControl(阴性对照)、miRNA(阳性对照)及对应的dsRNA至T24和EJ细胞,通过qPCR、RT-PCR和Western blot分别检测p21 mRNA和p21蛋白表达水平。结果:qPCR检测显示dsP21-245和dsP21-555均能明显促进两种细胞中p21mRNA水平的升高;与dsControl组相比,dsP21-245分别促进T24和EJ细胞中p21 mRNA的表达至2.32倍(P0.01)和2.84倍(P0.001);与miR-1236-3p组相比,dsP21-245分别在T24和EJ细胞中p21 mRNA表达的差异均没有统计学意义(P0.05);与dsControl组相比,dsP21-555均能明显促进T24和EJ细胞中p21 mRNA的高表达(P0.001,P0.001);与miR-370-5p组相比,dsP21-555分别在T24和EJ细胞中p21 mRNA表达的差异均没有统计学意义(P0.05)。通过RT-PCR得到了进一步验证。蛋白质印迹法提示,在T24和EJ细胞中,p21蛋白表达变化与p21 mRNA表达变化一致。与dsControl组相比,其余6对dsRNA均未能同时显著上调两种细胞系中p21~(WAF1)/CIP基因的表达(P0.05)。结论:外源性的dsP21-245和dsP21-555能显著促进膀胱癌细胞中p21~(WAF1)/CIP1的高表达,且与相应的内源性miRNA激活p21~(WAF1)/CIP1表达的能力无明显差异。第二部分外源性dsRNA通过激活p21蛋白的表达抑制膀胱癌细胞生长目的:通过转染外源性dsP21-555至膀胱癌细胞T24和EJ中,观察其能否抑制膀胱癌细胞的生长。方法:分别转染dsControl(阴性对照组)、miR-370-5p(阳性对照组)和dsP21-555(实验组)至膀胱癌细胞系T24和EJ。通过qPCR检测p21 mRNA和CDK4/6mRNA的表达变化。通过蛋白质印迹法检测p21蛋白和CDK4和CDK6蛋白的表达。通过流式细胞术检测三组中细胞周期分布。MTS法检测三组中细胞增殖能力。集落形成实验检测三组中单个细胞克隆增殖能力。结果:qPCR结果显示,与阴性对照组dsControl相比,dsP21-555分别促进T24和EJ细胞中p21 mRNA表达至2.46倍(P0.01)和2.60倍(P0.001);与miR-370-5p相比,T24和EJ细胞中p21 mRNA表达的差异均无统计学意义(P0.05);与阴性对照组dsControl相比,dsP21-555组T24和EJ细胞中CDK4mRNA的表达下调至0.57倍(P0.001)和0.46倍(P0.01),CDK6 mRNA的表达下调至0.61倍(P0.01)和0.64倍(P0.01);与miR-370-5p相比,T24和EJ细胞中CDK4和CDK6 mRNA表达的差异均没有统计学意义(P0.05)。蛋白质印迹法提示,p21和CDK4及CDK6蛋白表达变化与相应mRNA表达变化一致。FCM检测显示,与阴性对照组dsControl相比,转染miR-370-5p或dsP21-555后,处于G0/G1期的细胞比例明显上升,而处于S期和G2/M期的细胞比例下降,表明细胞周期被阻滞在G0/G1期。MTS法显示,与dsControl相比,转染miR-370-5p或dsP21-555后细胞增殖能力均明显减弱(P0.05),但与miR-370-5p相比,dsP21-555组细胞增殖能力无明显变化(P0.05)。细胞集落形成实验显示,阳对对照组miR-370-5p和实验组dsP21-555形成的集落数数量均较阴性对照组dsControl少。结论:外源性dsP21-555能激活p21蛋白的表达,具有对膀胱癌细胞生长的抑制作用。
[Abstract]:Part I comparison of the activation and expression of p21 protein in bladder cancer cells by exogenous and endogenous small molecule dsRNA: artificial synthesis of 8 pairs of small molecules dsRNA:dsP21-242, dsP21-243, dsP21-244, dsP21-245 and dsP21-552, dsP21-553, D, respectively, and the p21 gene promoter region of the p21 gene, respectively, and miR-370-5p, respectively. SP21-554, dsP21-555, respectively, to observe the differences in the /CIP1 expression of the tumor suppressor gene p21~ (WAF1) in human bladder cancer cell lines (T24 and EJ) with miR-1236-3p and miR-370-5p. Methods: 8 pairs of dsRNA were designed and synthesized by reference to the design principle of SA RNA. The expression level of p21 mRNA and p21 protein was detected by qPCR, RT-PCR and Western blot. Results: qPCR detection showed that dsP21-245 and dsP21-555 significantly promoted the increase of p21mRNA level in the two cells; compared with the dsControl group, it promoted the expression of 2.32 times and 2.84 times respectively. Compared with group R-1236-3p, there was no significant difference in the expression of p21 mRNA in T24 and EJ cells, respectively (P0.05). Compared with the dsControl group, dsP21-555 could significantly promote the high expression of p21 T24 and EJ cells in T24 and EJ cells. Statistical significance (P0.05) was further verified by RT-PCR. The expression of p21 protein in T24 and EJ cells was consistent with the changes of p21 mRNA expression in T24 and EJ cells. The expression of p21 ~ (WAF1) /CIP genes in the two cell lines was not significantly up-regulated at the same time as dsControl. SP21-245 and dsP21-555 can significantly promote the high expression of p21~ (WAF1) /CIP1 in bladder cancer cells, and no significant difference in the ability of corresponding endogenous miRNA to activate p21~ (WAF1) /CIP1 expression. The second part of exogenous dsRNA inhibits the growth of bladder cancer cells by activating the expression of p21 protein: transfection of exogenous dsP21-555 to bladder cancer cells by transfection In T24 and EJ, the growth of bladder cancer cells could be inhibited. Methods: transfection of dsControl (negative control group), miR-370-5p (positive control group) and dsP21-555 (experimental group) to T24 and EJ. of bladder cancer cell lines were used to detect the expression of p21 mRNA and CDK4/6mRNA by qPCR. The cell proliferation ability of three groups was detected by.MTS method in three groups by flow cytometry. Colony formation test was used to detect the proliferation ability of single cell clone in three groups. Results: qPCR results showed that dsP21-555 promoted p21 mRNA in T24 and EJ cells to 2.46 times (P0.01), respectively, compared with negative control group dsControl. 2.60 times (P0.001); compared with miR-370-5p, there was no significant difference in p21 mRNA expression in T24 and EJ cells (P0.05), and the expression of CDK4mRNA in T24 and EJ cells in the negative control group was down to 0.57 times and 0.46 times, and the expression was down to 0.61 times and 0.64 times. The differences in the expression of CDK4 and CDK6 mRNA in T24 and EJ cells were not statistically significant (P0.05). The difference between the expression of p21 and CDK4 and CDK6 protein was consistent with that of the corresponding mRNA expression, and the ratio of the cells to the negative control group was significantly higher than that of the negative control group. The percentage of cells in phase S and G2/M phase decreased, indicating that cell cycle was blocked in G0/G1 phase.MTS method, compared with dsControl, the cell proliferation ability of miR-370-5p or dsP21-555 decreased significantly (P0.05), but there was no significant change in cell proliferation ability of dsP21-555 group compared with miR-370-5p (P0.05). Cell colony formation experiment showed that The number of colony numbers of dsP21-555 in the control group miR-370-5p and the experimental group was less than that of the negative control group dsControl. Conclusion: exogenous dsP21-555 can activate the expression of p21 protein and have inhibitory effect on the growth of bladder cancer cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.14

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