本文選題：miR-1236-3p + miR-370-5p�。� 參考：《華中科技大學(xué)》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(duì)膀胱癌細(xì)胞中p21蛋白激活表達(dá)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(dòng)子序列區(qū)域完全互補(bǔ)配對(duì)的8對(duì)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(xì)胞系(T24和EJ)中抑癌基因p21~(WAF1)/CIP1表達(dá)能力的不同。方法:8對(duì)dsRNA通過(guò)參照sa RNA的設(shè)計(jì)原則設(shè)計(jì)合成。分別轉(zhuǎn)染dsControl(陰性對(duì)照)、miRNA(陽(yáng)性對(duì)照)及對(duì)應(yīng)的dsRNA至T24和EJ細(xì)胞,通過(guò)qPCR、RT-PCR和Western blot分別檢測(cè)p21 mRNA和p21蛋白表達(dá)水平。結(jié)果:qPCR檢測(cè)顯示dsP21-245和dsP21-555均能明顯促進(jìn)兩種細(xì)胞中p21mRNA水平的升高;與dsControl組相比,dsP21-245分別促進(jìn)T24和EJ細(xì)胞中p21 mRNA的表達(dá)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(xì)胞中p21 mRNA表達(dá)的差異均沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05);與dsControl組相比,dsP21-555均能明顯促進(jìn)T24和EJ細(xì)胞中p21 mRNA的高表達(dá)(P0.001,P0.001);與miR-370-5p組相比,dsP21-555分別在T24和EJ細(xì)胞中p21 mRNA表達(dá)的差異均沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05)。通過(guò)RT-PCR得到了進(jìn)一步驗(yàn)證。蛋白質(zhì)印跡法提示,在T24和EJ細(xì)胞中,p21蛋白表達(dá)變化與p21 mRNA表達(dá)變化一致。與dsControl組相比,其余6對(duì)dsRNA均未能同時(shí)顯著上調(diào)兩種細(xì)胞系中p21~(WAF1)/CIP基因的表達(dá)(P0.05)。結(jié)論:外源性的dsP21-245和dsP21-555能顯著促進(jìn)膀胱癌細(xì)胞中p21~(WAF1)/CIP1的高表達(dá),且與相應(yīng)的內(nèi)源性miRNA激活p21~(WAF1)/CIP1表達(dá)的能力無(wú)明顯差異。第二部分外源性dsRNA通過(guò)激活p21蛋白的表達(dá)抑制膀胱癌細(xì)胞生長(zhǎng)目的:通過(guò)轉(zhuǎn)染外源性dsP21-555至膀胱癌細(xì)胞T24和EJ中,觀察其能否抑制膀胱癌細(xì)胞的生長(zhǎng)。方法:分別轉(zhuǎn)染dsControl(陰性對(duì)照組)、miR-370-5p(陽(yáng)性對(duì)照組)和dsP21-555(實(shí)驗(yàn)組)至膀胱癌細(xì)胞系T24和EJ。通過(guò)qPCR檢測(cè)p21 mRNA和CDK4/6mRNA的表達(dá)變化。通過(guò)蛋白質(zhì)印跡法檢測(cè)p21蛋白和CDK4和CDK6蛋白的表達(dá)。通過(guò)流式細(xì)胞術(shù)檢測(cè)三組中細(xì)胞周期分布。MTS法檢測(cè)三組中細(xì)胞增殖能力。集落形成實(shí)驗(yàn)檢測(cè)三組中單個(gè)細(xì)胞克隆增殖能力。結(jié)果:qPCR結(jié)果顯示,與陰性對(duì)照組dsControl相比,dsP21-555分別促進(jìn)T24和EJ細(xì)胞中p21 mRNA表達(dá)至2.46倍(P0.01)和2.60倍(P0.001);與miR-370-5p相比,T24和EJ細(xì)胞中p21 mRNA表達(dá)的差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);與陰性對(duì)照組dsControl相比,dsP21-555組T24和EJ細(xì)胞中CDK4mRNA的表達(dá)下調(diào)至0.57倍(P0.001)和0.46倍(P0.01),CDK6 mRNA的表達(dá)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(xì)胞中CDK4和CDK6 mRNA表達(dá)的差異均沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05)。蛋白質(zhì)印跡法提示,p21和CDK4及CDK6蛋白表達(dá)變化與相應(yīng)mRNA表達(dá)變化一致。FCM檢測(cè)顯示,與陰性對(duì)照組dsControl相比,轉(zhuǎn)染miR-370-5p或dsP21-555后,處于G0/G1期的細(xì)胞比例明顯上升,而處于S期和G2/M期的細(xì)胞比例下降,表明細(xì)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(xì)胞增殖能力均明顯減弱(P0.05),但與miR-370-5p相比,dsP21-555組細(xì)胞增殖能力無(wú)明顯變化(P0.05)。細(xì)胞集落形成實(shí)驗(yàn)顯示,陽(yáng)對(duì)對(duì)照組miR-370-5p和實(shí)驗(yàn)組dsP21-555形成的集落數(shù)數(shù)量均較陰性對(duì)照組dsControl少。結(jié)論:外源性dsP21-555能激活p21蛋白的表達(dá),具有對(duì)膀胱癌細(xì)胞生長(zhǎng)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(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R737.14

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本文編號(hào)：1929801