Mir-29c在膀胱癌组织中表达降低并抑制膀胱癌细胞增殖

发布时间：2018-05-26 02:18

本文选题：MiR-29c + 膀胱移行细胞癌　；参考：《重庆医科大学》2014年硕士论文

【摘要】：目的：miR-29c在膀胱癌组织及癌旁组织中的差异表达及其与临床各病理参数的关系。方法：利用实时荧光定量PCR检测30例膀胱癌组织及癌旁组织中miR-29c的表达；Ta期：5例，T1期：15例，T2-4期：10例，参照miR-29c在膀胱癌组织中表达的中位数（0.3496），将其分为2组：低表达组15例和高表达组15例，，分析其与临床各病理参数之间的关系结果：膀胱癌组织中miR-29c表达量明显低于癌旁组织（P0.01）,miR-29c低表达与肿瘤的分期有关（P0.01）。结论：MiR-29c在膀胱癌组织中表达下调,可能与膀胱癌的分期有关。目的：构建miRNA-29a/c的重组腺病毒并在膀胱癌T24细胞中坚定。方法：以人全基因组DNA为模板, PCR扩增miR-29a、miR-29c,克隆至腺病毒穿梭载体pAdtrace-TO4-CMV。重组穿梭载体经pmeI线性化后与腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌BJ5183,通过同源重组获得重组腺病毒质粒pAdEasy-1-miR-29a、pAdEasy-1-miR-29c, pac I线性化后转染HEK-293细胞,进行包装和扩增。实时荧光定量PCR检测感染腺病毒的膀胱癌T24细胞中miR-29a、miR-29c的表达水平。结果：经DNA测序和限制性内切酶分析显示,重组腺病毒质粒pAdEasy-1-miR-29a、 pAdEasy-1-miR-29c构建成功;感染腺病毒Ad-miR-29a和Ad-miR-29c后,经实时荧光定量PCR检测,膀胱癌细胞中miR-29a、miR-29c表达显著增高(P0.01)。结论：已成功构建miR-29a、miR-29c腺病毒,为研究其在膀胱癌细胞的生物学功能奠定基础。目的：体外研究miR-29c生物学作用及其抑制膀胱移形细胞癌增殖的分子机制。方法：用CCK-8检测T24细胞增殖能力，FCM检测T24细胞周期分布及细胞凋亡，transwell实验检测细胞的迁移能力。Western blot检测AKT/GSK3β通路中增殖相关蛋白的表达。结果：过表达miR-29c显著抑制膀胱癌细胞T24的增殖（P0.01）、迁移（P0.05）, T24细胞阻滞于G0/G1期（P0.01）并能促进细胞凋亡（P0.01）. Western blot结果显示：与对照组相比过表达miR-29c组中c-myc蛋白表达量显著降低（P0.01）;而cyclinD1蛋白的表达则无明显变化。miR-29c+LY294002组比单独使用miR-29c组和LY294002组p-AKT和p-GSK3β蛋白表达显著降低（P0.01）结论：miR-29c可能通过AKT-GSK3β pathway抑制T24细胞的增殖，其在膀胱癌的发展中起着重要的作用。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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