MicroRNA-200b调控膀胱癌发生EMT与Notch信号通路关系的实验研究

发布时间：2018-07-05 11:39

本文选题：膀胱癌 + Notch信号通路　；参考：《南昌大学》2014年硕士论文

【摘要】：目的：探讨microRNA-200b调控膀胱癌发生EMT与Notch信号通路关系的内在分子机制。方法：将has-miR-200b mimics及阴性对照组(scramble)用脂质体Lip2000转染膀胱癌T24、5637细胞，通过倒置显微镜观察膀胱癌T24、5637细胞生长及形态的变化；采用qRT-PCR检测转染效率；运用RT-PCR及western blot技术检测E-cadherin、N-cadherin、vimentin、alpha-smooth muscle actin、Notch-1、Jagged-1的mRNA及蛋白表达变化；MTT检测转染后膀胱癌细胞株耐药性的改变；采用Hoechst染色法检测抗凋亡能力的改变；细胞凋亡率的改变通过流式细胞学检测；转染后的膀胱癌细胞株侵袭能力的变化运用transwell来评估。结果：实验组的膀胱癌细胞数量变少，分布更分散，细胞的体积明显增大，形态更加不规则；qRT-PCR结果示：转染48h后，miR-200b显著性上调（P＜0.05）；RT-PCR及western blot结果表明：E-cadherin mRNA和蛋白表达显著上调（P＜0.05），N-cadherin、vimentin、alpha-smooth muscle actin、Notch-1、Jagged-1mRNA和蛋白显著下调（P＜0.05）；MTT结果提示:实验组细胞对顺铂的抵抗力降低（P＜0.05）；Hoechst33342表明:实验组的膀胱癌细胞株抗凋亡能力减弱（P＜0.05）；流式细胞学结果示：实验组的早期晚期凋亡率显著增加，差异均具有统计学意义（P＜0.05）；Transwell表明实验组膀胱癌细胞的侵袭能力明显下调（P＜0.05）。结论：上调microRNA-200b能通过抑制Notch-1、Jagged-1的mRNA及蛋白表达，，从而阻断Notch信号通路，进而抑制膀胱癌发生EMT；同时对膀胱癌细胞的耐药性、抗凋亡能力、侵袭性产生影响。
[Abstract]:Aim: to investigate the molecular mechanism of microRNA-200b regulating the relationship between EMT and Notch signaling pathway in bladder cancer. Methods: has-miR-200b mimics and negative control group (scramble) were transfected with liposome Lip2000 into bladder cancer cell line T2465637. The growth and morphology of T24567 cells were observed by inverted microscope, and the transfection efficiency was detected by qRT-PCR. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to detect the changes of mRNA and protein expression of E-cadherin N-cadherin alpha-smooth muscle actinus Notch-1 and Jagged-1. The changes of drug resistance of bladder cancer cell lines after transfection were detected by MTT assay, the changes of anti-apoptosis ability were detected by Hoechst staining, and the changes of apoptosis rate were detected by flow cytometry. Changes in invasive ability of bladder cancer cell lines after transfection were evaluated by transwell. Results: in the experimental group, the number of bladder cancer cells decreased, the distribution became more dispersed, the volume of the cells increased significantly, and the morphology became more irregular. The results of qRT-PCR showed that miR-200b was significantly up-regulated 48 hours after transfection (P < 0.05). The results of RT-PCR and western blot showed that the expression of E-cadherin mRNA and protein was significantly up-regulated (P < 0. 05). (P < 0. 05). The results of western blot showed that the resistance to cisplatin was decreased in the experimental group (P < 0. 05), and the expression of Jagged-1 mRNA and protein was significantly down-regulated (P < 0. 05). Hoechst33342 showed that the anti-apoptotic ability of bladder cancer cell line in the experimental group was decreased (P < 0.05), and the apoptosis rate in the early and late stage of the experimental group was significantly increased (P < 0.05), and the apoptosis rate in the early and late stage of the experimental group was significantly higher than that in the control group (P < 0.05). Transwell showed that the invasion ability of bladder cancer cells in the experimental group was significantly down-regulated (P < 0.05). Conclusion: upregulation of microRNA-200b can block Notch signaling pathway by inhibiting the expression of Notch-1 and Jagged-1 mRNA and protein, and then inhibit the development of EMTs in bladder cancer, and affect the resistance, anti-apoptosis and invasiveness of bladder cancer cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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