沉默钟基因PER2对人口腔鳞癌SCC15细胞生物学行为的影响及机制

发布时间：2018-03-17 07:38

本文选题：肿瘤　切入点：口腔癌　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究生物钟基因PER2对人类口腔鳞癌SCC15细胞凋亡、增殖、侵袭、迁移的影响与机理。方法:利用RNAi技术构建出3组靶向PER2基因shRNA慢病毒质粒,感染人类口腔鳞状细胞癌SCC15细胞;经过测序、实时荧光定量PCR以及Western blot检测方法鉴定并筛选出PER2基因沉默效果最佳组为实验组,对照组为不包含PER2片段的scramble质粒病毒,空白组为未经过任何的处理的SCC15细胞。应用实时荧光定量PCR检测Ki-67、MDM2、P53、Bcl-2、Bax、c-Myc、MMP-2、Timp-2和VEGFmRNA的表达改变;CCK-8试验用于检测SCC15细胞体外生长能力,流式细胞仪用于检测基因沉默后细胞增殖、凋亡水平、细胞周期分布,平板克隆形成实验用于检测细胞克隆形成率,Transwell小室用于检测细胞迁移、侵袭能力改变,裸鼠的体内成瘤实验用于检测人口腔鳞癌细胞SCC15体内的成瘤情况。结果:成功的构建了3组靶向PER2基因的PER2-shRNA慢病毒质粒,PER2-shRNA-I组沉默效果为最佳,即被作为实验组。沉默PER2基因后,Ki-67、MDM2、Bcl-2、c-Myc、MMP-2和VEGF mRNA的表达水平显著升高(均P0.05),p53、Bax和Timp-2 mRNA的表达水平显著降低(均P0.05)。PER2沉默后,SCC15细胞的生长能力、细胞增殖指数、细胞迁移和侵袭能力显著升高(均P0.05),凋亡指数显著降低(P0.05)。体内实验也证明PER2沉默后SCC15细胞的成瘤能力显著增强(P0.05)。结论:表明钟基因PER2通过对下游许多重要肿瘤相关基因调控而发挥重要抑癌作用,对生物钟PER2基因的深入研究有望成为治疗癌症的新分子靶点。
[Abstract]:Objective: to study the effect and mechanism of clock gene PER2 on apoptosis, proliferation, invasion and migration of human oral squamous cell carcinoma (SCC15) cells. Methods: three groups of shRNA lentivirus plasmids targeting PER2 gene were constructed by RNAi technique. SCC15 cells infected with human oral squamous cell carcinoma were identified and screened by sequencing, real-time fluorescence quantitative PCR and Western blot detection methods. The best silencing effect of PER2 gene was identified as experimental group, and the control group was scramble plasmid virus without PER2 fragment. The expression changes of Ki-67M2MDM2P53P53Bcl-2Bax-2MP-2Timp-2 and VEGFmRNA were detected by flow cytometry. Flow cytometry was used to detect the proliferation and apoptosis of SCC15 cells after gene silencing, and CCK-8 assay was used to detect the proliferation and apoptosis of SCC15 cells after gene silencing. Cell cycle distribution, flat plate clone formation assay was used to detect cell clone formation rate and Transwell chamber was used to detect cell migration and invasion ability. Results: three groups of PER2-shRNA lentivirus plasmids targeting PER2 gene, PER2-shRNA-I, were successfully constructed, and the silencing effect of PER2-shRNA-I group was the best. After silencing the PER2 gene, the expression levels of Ki-67m2m2mcl-2c-Mycnc-MMP-2 and VEGF mRNA were significantly increased (both P0.05P53BX and Timp-2 mRNA were significantly decreased after silencing the PER2 gene (all P0.05PER2) after silencing SCC15 cells, the growth ability and proliferation index of SCC15 cells were significantly decreased. The ability of cell migration and invasion was significantly increased (all P0.05N, apoptotic index significantly decreased P0.050.The in vivo experiments also proved that the tumorigenic ability of SCC15 cells after PER2 silencing was significantly enhanced. Conclusion: it is suggested that the clock gene PER2 can significantly enhance the tumorigenesis of SCC15 cells in many important tumor phases downstream. Gene regulation plays an important role in the inhibition of cancer, Further study on PER2 gene of biological clock is expected to be a new molecular target for cancer treatment.
【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】：R739.8

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