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  本文選題：MiR-29c + 膀胱移行細胞癌�。� 參考：《重慶醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：miR-29c在膀胱癌組織及癌旁組織中的差異表達及其與臨床各病理參數(shù)的關(guān)系。 方法：利用實時熒光定量PCR檢測30例膀胱癌組織及癌旁組織中miR-29c的表達；Ta期：5例，T1期：15例，T2-4期：10例，參照miR-29c在膀胱癌組織中表達的中位數(shù)（0.3496），將其分為2組：低表達組15例和高表達組15例，，分析其與臨床各病理參數(shù)之間的關(guān)系 結(jié)果：膀胱癌組織中miR-29c表達量明顯低于癌旁組織（P0.01）,miR-29c低表達與腫瘤的分期有關(guān)（P0.01）。 結(jié)論：MiR-29c在膀胱癌組織中表達下調(diào),可能與膀胱癌的分期有關(guān)。 目的：構(gòu)建miRNA-29a/c的重組腺病毒并在膀胱癌T24細胞中堅定。 方法：以人全基因組DNA為模板, PCR擴增miR-29a、miR-29c,克隆至腺病毒穿梭載體pAdtrace-TO4-CMV。重組穿梭載體經(jīng)pmeI線性化后與腺病毒骨架質(zhì)粒pAdEasy-1共轉(zhuǎn)化感受態(tài)大腸桿菌BJ5183,通過同源重組獲得重組腺病毒質(zhì)粒pAdEasy-1-miR-29a、pAdEasy-1-miR-29c, pac I線性化后轉(zhuǎn)染HEK-293細胞,進行包裝和擴增。實時熒光定量PCR檢測感染腺病毒的膀胱癌T24細胞中miR-29a、miR-29c的表達水平。 結(jié)果：經(jīng)DNA測序和限制性內(nèi)切酶分析顯示,重組腺病毒質(zhì)粒pAdEasy-1-miR-29a、 pAdEasy-1-miR-29c構(gòu)建成功;感染腺病毒Ad-miR-29a和Ad-miR-29c后,經(jīng)實時熒光定量PCR檢測,膀胱癌細胞中miR-29a、miR-29c表達顯著增高(P0.01)。 結(jié)論：已成功構(gòu)建miR-29a、miR-29c腺病毒,為研究其在膀胱癌細胞的生物學(xué)功能奠定基礎(chǔ)。 目的：體外研究miR-29c生物學(xué)作用及其抑制膀胱移形細胞癌增殖的分子機制。 方法：用CCK-8檢測T24細胞增殖能力，F(xiàn)CM檢測T24細胞周期分布及細胞凋亡，transwell實驗檢測細胞的遷移能力。Western blot檢測AKT/GSK3β通路中增殖相關(guān)蛋白的表達。 結(jié)果：過表達miR-29c顯著抑制膀胱癌細胞T24的增殖（P0.01）、遷移（P0.05）, T24細胞阻滯于G0/G1期（P0.01）并能促進細胞凋亡（P0.01）. Western blot結(jié)果顯示：與對照組相比過表達miR-29c組中c-myc蛋白表達量顯著降低（P0.01）;而cyclinD1蛋白的表達則無明顯變化。miR-29c+LY294002組比單獨使用miR-29c組和LY294002組p-AKT和p-GSK3β蛋白表達顯著降低（P0.01） 結(jié)論：miR-29c可能通過AKT-GSK3β pathway抑制T24細胞的增殖，其在膀胱癌的發(fā)展中起著重要的作用。
[Abstract]:Objective to investigate the differential expression of miR-29c in bladder cancer and its relationship with clinicopathologic parameters. Methods: real-time fluorescence quantitative PCR was used to detect the expression of miR-29c in 30 cases of bladder cancer and its adjacent tissues. According to the median expression of miR-29c in bladder cancer tissue, it was divided into two groups: low expression group (15 cases) and high expression group (15 cases). The relationship between miR-29c and clinicopathologic parameters was analyzed. Results: the expression of miR-29c in bladder cancer was significantly lower than that in adjacent tissues. The low expression of P0.01miR-29c was related to the stage of bladder cancer. Conclusion the down-regulation of the expression of MiR-29c in bladder cancer may be related to the stage of bladder cancer. Objective: to construct recombinant adenovirus of miRNA-29a/c and be firm in bladder cancer T 24 cells. Methods: using human genomic DNA as template, miR-29ahmiR-29cwas amplified by PCR and cloned into adenovirus shuttle vector pAdtrace-TO4-CMV. The recombinant shuttle vector was linearized by pmeI and co-transformed with adenovirus skeleton plasmid pAdEasy-1 into competent Escherichia coli BJ5183.Recombinant adenovirus plasmid pAdEasy-1-miR-29a- pAdEasy-1-miR-29c. pac I was linearized and transfected into HEK-293 cells for packaging and amplification. The expression of miR-29a- miR-29c in bladder cancer T24 cells infected with adenovirus was detected by real-time fluorescence quantitative PCR. Results: DNA sequencing and restriction endonuclease analysis showed that the recombinant adenovirus plasmid pAdEasy-1-miR-29a was successfully constructed, and the expression of miR-29c in bladder cancer cells was significantly increased by real-time fluorescence quantitative PCR detection after infection with Ad-miR-29a and Ad-miR-29c. Conclusion: miR-29a miR-29c adenovirus has been successfully constructed, which lays a foundation for the study of its biological function in bladder cancer cells. Objective: to study the biological effect of miR-29c and its molecular mechanism in inhibiting the proliferation of bladder carcinoma. Methods: the proliferation ability of T24 cells was detected by CCK-8 and the cell cycle distribution was detected by FCM. The migration ability of T24 cells was detected by apoptosis transwell assay. The expression of proliferation-associated protein in AKT/GSK3 尾 pathway was detected by Western blot. Results: overexpression of miR-29c significantly inhibited the proliferation of bladder cancer cell line T24 (P0.01A), and the migration of T24 cells was blocked in G0/G1 phase (P0.01) and promoted the apoptosis of T24 cells. The results of Western blot showed that the expression of c-myc protein in miR-29c group was significantly lower than that in control group, while the expression of cyclinD1 protein in LY294002 group was not significantly different from that in miR-29c group and LY294002 group, and the expression of p-AKT and p-GSK3 尾 protein in miR-29c group was significantly lower than that in LY294002 group (P 0.01). ConclusionMiR-29c may inhibit the proliferation of T24 cells through AKT-GSK3 尾 pathway, which plays an important role in the development of bladder cancer.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.14

【參考文獻】

相關(guān)期刊論文 前1條

1 Binhua P.Zhou;;Epithelial-mesenchymal transition in breast cancer progression and metastasis[J];癌癥;2011年09期

本文編號：1935559