DEPDC1稳定沉默CNE-1细胞株的构建与分析

发布时间：2018-03-11 12:18

本文选题：鼻咽癌　切入点：DEPDC1　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：DEPDC1(DEP domain containing 1)是近年来发现的一个肿瘤相关基因,前期研究结果表明DEPDC1在鼻咽癌细胞周期以及癌变过程中具有重要作用。目的:为了更进一步探讨其在鼻咽癌发生发展中的作用。方法:利用qRT-PCR和免疫组织化学染色分别检测DEPDC1在33例新鲜鼻咽癌组织样品和44例鼻咽癌组织芯片中的表达情况。人鼻咽癌细胞株CNE-1稳定沉默DEPDC1后,流式细胞技术检测细胞周期,CCK8检测细胞增殖,transwell分析细胞侵袭迁移的情况,利用裸鼠成瘤试验分析体内成瘤能力。结果:DEPDC1在鼻咽癌组织中表达量(0.699±0.521)明显高于鼻咽部正常组织(0.408±0.183),差异有统计学意义(P0.05)。DEPDC1稳定沉默后,CCK8检测结果表明CNE-1细胞增殖速度减慢;流式细胞检测结果显示,G1期细胞减少,G2/M期细胞增多,细胞周期进程受阻。划痕及Transwell实验结果表明,DEPDC1稳定沉默明显减弱了CNE-1细胞运动、侵袭及迁移能力(77.033±5.183 20.137±2.828)(107.336±5.033 26.326±1.414),差异均有统计学意义(P0.05)。进一步定量qRT-PCR及Western印迹检测发现,DEPDC1稳定沉默导致上皮细胞标志分子Ecadherin(0.457±0.022)显著上调、而间质细胞标志分子Vimentin(0.780±0.063)、Ncadherin(0.780±0.063)以及EMT上游关键转录因子Twist1(0.710±0.034)下调(P0.05)。裸鼠成瘤实验表明DEPDC1稳定沉默后抑制了裸鼠成瘤能力,与对照组(0.23±0.03g,46.91±15.07mm3)相比,DEPDC1稳定沉默后肿瘤重量和体积(0.56±0.17g,546.24±93.46mm3)明显减小,差异均有统计学意义(P0.05)。结论:本研究成功构建了DEPDC1稳定沉默的鼻咽癌细胞株,与前期siRNA介导的瞬时沉默相似,DEPDC1稳定沉默可抑制鼻咽癌细胞的增殖、侵袭迁移,并改变EMT关键分子的表达,为进一步探索DEPDC1在鼻咽癌中的作用机理奠定了基础。
[Abstract]:DEPDC1(DEP domain containing 1 is a tumor-related gene discovered in recent years. Previous studies have shown that DEPDC1 plays an important role in the cell cycle and carcinogenesis of nasopharyngeal carcinoma. Objective: to further explore the role of DEPDC1 in the carcinogenesis and development of nasopharyngeal carcinoma. Methods: qRT-PCR and immunohistochemical staining were used to study the role of DEPDC1 in the development of nasopharyngeal carcinoma. The expression of DEPDC1 in 33 fresh nasopharyngeal carcinoma tissue samples and 44 nasopharyngeal carcinoma tissue microarray samples were detected respectively. Human nasopharyngeal carcinoma cell line CNE-1 was stably silenced with DEPDC1. Flow cytometric analysis of cell cycle CCK8 cell proliferation and transwell analysis of cell invasion and migration. Results the expression of w DEPDC1 in nasopharyngeal carcinoma was 0.699 卤0.521), which was significantly higher than that in normal nasopharyngeal tissue (0.408 卤0.183). The results showed that the proliferation rate of CNE-1 cells slowed down after stable silencing. The results of flow cytometry showed that the G 1 phase cells decreased the number of G 2 / M phase cells and the cell cycle progression was blocked. The results of scratch and Transwell showed that the stable silencing of CNE-1 cells significantly decreased the motility of CNE-1 cells. The ability of invasion and migration was 77.033 卤5.183 20.137 卤2.828 (107.336 卤5.033 26.326 卤1.414), the difference was statistically significant (P 0.05). Further quantitative qRT-PCR and Western blot analysis showed that stable silencing of DEPDC1 resulted in a marked up-regulation of Ecadherin(0.457 卤0.022 in epithelial cells. However, Vimentin(0.780 卤0.063 + Ncadherin (0.780 卤0.063) and Twist1(0.710 卤0.034 (a key transcription factor in upstream of EMT) down-regulated P0.05.The tumor-forming ability of nude mice was inhibited by DEPDC1 stable silencing, and the tumor weight and volume of DEPDC1 decreased significantly after stable silencing compared with control group (0.23 卤0.03g / L, 46.91 卤15.073mm), and the tumor weight and volume decreased significantly (0.56 卤0.17g / 546.24 卤93.46mm3), compared with that of the control group (0.23 卤0.063 g / L, 46.91 卤15.073mm). Conclusion: DEPDC1 stable silencing nasopharyngeal carcinoma cell line was successfully constructed. Similar to the transient silencing mediated by siRNA, DEPDC1 stable silencing can inhibit the proliferation, invasion and migration of nasopharyngeal carcinoma cells. The expression of key molecules of EMT was changed, which laid a foundation for further exploring the mechanism of DEPDC1 in nasopharyngeal carcinoma.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.63

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