本文選題：miR-1236-3p + miR-370-5p　； 參考：《華中科技大學》2016年碩士論文

【摘要】：第一部分外源性和內(nèi)源性小分子dsRNA對膀胱癌細胞中p21蛋白激活表達能力的比較目的:人工合成分別與miR-1236-3p、miR-370-5p所作用的p21基因啟動子序列區(qū)域完全互補配對的8對小分子dsRNA:dsP21-242、dsP21-243、dsP21-244、dsP21-245和dsP21-552、dsP21-553、dsP21-554、dsP21-555,分別觀察其與miR-1236-3p、miR-370-5p在上調(diào)人膀胱癌細胞系(T24和EJ)中抑癌基因p21~(WAF1)/CIP1表達能力的不同。方法:8對dsRNA通過參照sa RNA的設(shè)計原則設(shè)計合成。分別轉(zhuǎn)染dsControl(陰性對照)、miRNA(陽性對照)及對應(yīng)的dsRNA至T24和EJ細胞,通過qPCR、RT-PCR和Western blot分別檢測p21 mRNA和p21蛋白表達水平。結(jié)果:qPCR檢測顯示dsP21-245和dsP21-555均能明顯促進兩種細胞中p21mRNA水平的升高;與dsControl組相比,dsP21-245分別促進T24和EJ細胞中p21 mRNA的表達至2.32倍(P0.01)和2.84倍(P0.001);與miR-1236-3p組相比,dsP21-245分別在T24和EJ細胞中p21 mRNA表達的差異均沒有統(tǒng)計學意義(P0.05);與dsControl組相比,dsP21-555均能明顯促進T24和EJ細胞中p21 mRNA的高表達(P0.001,P0.001);與miR-370-5p組相比,dsP21-555分別在T24和EJ細胞中p21 mRNA表達的差異均沒有統(tǒng)計學意義(P0.05)。通過RT-PCR得到了進一步驗證。蛋白質(zhì)印跡法提示,在T24和EJ細胞中,p21蛋白表達變化與p21 mRNA表達變化一致。與dsControl組相比,其余6對dsRNA均未能同時顯著上調(diào)兩種細胞系中p21~(WAF1)/CIP基因的表達(P0.05)。結(jié)論:外源性的dsP21-245和dsP21-555能顯著促進膀胱癌細胞中p21~(WAF1)/CIP1的高表達,且與相應(yīng)的內(nèi)源性miRNA激活p21~(WAF1)/CIP1表達的能力無明顯差異。第二部分外源性dsRNA通過激活p21蛋白的表達抑制膀胱癌細胞生長目的:通過轉(zhuǎn)染外源性dsP21-555至膀胱癌細胞T24和EJ中,觀察其能否抑制膀胱癌細胞的生長。方法:分別轉(zhuǎn)染dsControl(陰性對照組)、miR-370-5p(陽性對照組)和dsP21-555(實驗組)至膀胱癌細胞系T24和EJ。通過qPCR檢測p21 mRNA和CDK4/6mRNA的表達變化。通過蛋白質(zhì)印跡法檢測p21蛋白和CDK4和CDK6蛋白的表達。通過流式細胞術(shù)檢測三組中細胞周期分布。MTS法檢測三組中細胞增殖能力。集落形成實驗檢測三組中單個細胞克隆增殖能力。結(jié)果:qPCR結(jié)果顯示,與陰性對照組dsControl相比,dsP21-555分別促進T24和EJ細胞中p21 mRNA表達至2.46倍(P0.01)和2.60倍(P0.001);與miR-370-5p相比,T24和EJ細胞中p21 mRNA表達的差異均無統(tǒng)計學意義(P0.05);與陰性對照組dsControl相比,dsP21-555組T24和EJ細胞中CDK4mRNA的表達下調(diào)至0.57倍(P0.001)和0.46倍(P0.01),CDK6 mRNA的表達下調(diào)至0.61倍(P0.01)和0.64倍(P0.01);與miR-370-5p相比,T24和EJ細胞中CDK4和CDK6 mRNA表達的差異均沒有統(tǒng)計學意義(P0.05)。蛋白質(zhì)印跡法提示,p21和CDK4及CDK6蛋白表達變化與相應(yīng)mRNA表達變化一致。FCM檢測顯示,與陰性對照組dsControl相比,轉(zhuǎn)染miR-370-5p或dsP21-555后,處于G0/G1期的細胞比例明顯上升,而處于S期和G2/M期的細胞比例下降,表明細胞周期被阻滯在G0/G1期。MTS法顯示,與dsControl相比,轉(zhuǎn)染miR-370-5p或dsP21-555后細胞增殖能力均明顯減弱(P0.05),但與miR-370-5p相比,dsP21-555組細胞增殖能力無明顯變化(P0.05)。細胞集落形成實驗顯示,陽對對照組miR-370-5p和實驗組dsP21-555形成的集落數(shù)數(shù)量均較陰性對照組dsControl少。結(jié)論:外源性dsP21-555能激活p21蛋白的表達,具有對膀胱癌細胞生長的抑制作用。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R737.14

【參考文獻】

相關(guān)期刊論文 前2條

1 陳忠;;小分子非編碼RNA基因激活研究進展[J];現(xiàn)代泌尿生殖腫瘤雜志;2014年01期

2 陳忠;李龍承;;RNA激活研究進展[J];現(xiàn)代泌尿生殖腫瘤雜志;2012年02期

，

本文編號：1929801