食管鳞癌中miR-199a-3p对细胞增殖、凋亡的影响及分子机制研究

发布时间：2018-05-28 05:48

本文选题：食管鳞癌 + miR-199a-3p　；参考：《郑州大学》2017年硕士论文

【摘要】：研究目的1.探讨miR-199a-3p对食管鳞癌细胞增殖和凋亡的影响及可能的分子机制。2.探讨下调Rictor表达后PI3K/AKT/m TOR通路抑制剂对食管鳞癌细胞增殖和凋亡影响的变化情况。研究方法1.q RT-PCR法检测miR-199a-3p在五株食管鳞癌细胞TE1、ECa109、EC9706、KYSE450、KYSE790(其中TE1和EC9706属于低分化细胞株,ECa109、KYSE450和KYSE790属于高分化细胞株)中的表达情况。2.利用si RNA-MateTM转染试剂将人工合成的miR-199a-3p的模拟物mimics转染至食管鳞癌细胞ECa109、EC9706、KYSE450中,q RT-PCR法分别检测转染前后miR-199a-3p在三株细胞中的表达情况。分别采用克隆形成实验、CCK-8法及流式细胞术检测转染前后miR-199a-3p对食管鳞癌细胞增殖及凋亡的影响。3.分别用Western blots和q RT-PCR法检测转染前后三株食管鳞癌细胞中m TORC1信号通路相关因子m TOR、Raptor、p70S6K蛋白及m RNA的表达情况,凋亡相关蛋白Bcl-2以及自噬相关蛋白p62和LC3I/II的表达情况。4.用m TORC1抑制剂RAD001(30μM)处理三株食管鳞癌细胞,q RT-PCR法检测加药前后三株细胞中miR-199a-3p的表达情况。5.用PI3K/AKT/m TOR通路抑制剂处理筛选的稳定表达Rictor-sh RNA载体的ECa109细胞及未转染细胞,分别采用克隆形成实验和流式细胞术检测下调Rictor表后细胞增殖和凋亡的变化情况。结果1.miR-199a-3p在五株食管鳞癌细胞中均有表达但其表达水平无显著性差异,说明其表达与细胞分化程度无明显相关性。2.与未转染组和转染miR-199a-3p mimics阴性对照组的细胞相比,转染miR-199a-3p mimics的三株食管鳞癌细胞中miR-199a-3p的表达水平均显著升高,且三株细胞的克隆形成能力显著降低,细胞增殖明显减慢,细胞凋亡数显著增加。3.三株食管鳞癌细胞中,与未转染组和转染miR-199a-3p mimics阴性对照的细胞相比,转染miR-199a-3p mimics的细胞中m TOR、Raptor、p70S6K的蛋白和m RNA表达水平均显著降低,抗凋亡蛋白Bcl-2的表达水平显著降低,自噬蛋白p62表达水平明显降低,LC3I/II蛋白表达水平明显升高。4.三株食管鳞癌细胞中,与对照组相比,加RAD001的细胞中miR-199a-3p表达量无显著性差异。5.食管鳞癌ECa109细胞中,与未处理组相比,Rictor表达下调的细胞用PI3K/AKT/m TOR通路抑制剂处理后,细胞克隆形成数均明显减少,细胞凋亡数均明显增加。结论1.miR-199a-3p能够抑制食管鳞癌细胞增殖,诱导细胞凋亡,其机制可能与m TORC1/p70S6K信号通路和细胞自噬有关。2.下调Rictor表达后能增强PI3K/AKT/m TOR信号通路抑制剂对细胞增殖及凋亡的作用效果。
[Abstract]:Objective 1. to investigate the effect of miR-199a-3p on the proliferation and apoptosis of esophageal squamous cell carcinoma cells and the possible molecular mechanism.2. to investigate the changes of the effect of PI3K/AKT/m TOR pathway inhibitor on the proliferation and apoptosis of esophageal squamous cell carcinoma cells after the downregulation of Rictor. The 1.q RT-PCR method was used to detect TE1, ECa109, EC of miR-199a-3p in five esophageal squamous cell carcinoma cells. 9706, KYSE450, KYSE790 (of which TE1 and EC9706 belong to low differentiated cell lines, ECa109, KYSE450 and KYSE790 belong to highly differentiated cell lines).2. using Si RNA-MateTM transfection reagent to transfect the synthetic miR-199a-3p analogue mimics to esophageal squamous cell carcinoma cells. The expression of miR-199a-3p in three cells. The effects of miR-199a-3p on the proliferation and apoptosis of esophageal squamous cell carcinoma were detected by clones, CCK-8 and flow cytometry, and.3. was used to detect m TORC1 signaling pathway related factor M TOR in three esophageal squamous cell carcinoma cells before and after transfection, respectively. Expression of ptor, p70S6K protein and m RNA, apoptosis related protein Bcl-2, and expression of autophagy related protein p62 and LC3I/II.4. using M TORC1 inhibitor RAD001 (30 micron M) to treat three esophageal squamous cell carcinoma cells. The ECa109 cells and untransfected cells expressed the Rictor-sh RNA carrier, and the cell proliferation and apoptosis were detected by clone formation and flow cytometry. The results showed that 1.miR-199a-3p was expressed in five esophageal squamous cell carcinoma cells, but the expression was not significantly different, indicating the expression and cell expression of the cells. There was no significant correlation between the degree of differentiation and the expression of miR-199a-3p in three esophageal squamous cell carcinoma cells transfected with miR-199a-3p mimics, compared with those in the untransfected group and the cells transfected with miR-199a-3p mimics negative control group, and the expression level of miR-199a-3p was significantly increased in three cells transfected with miR-199a-3p mimics, and the clone formation ability of the cells was significantly reduced, the cell proliferation slowed down, and the cell apoptosis was significant. The expression level of M TOR, Raptor, p70S6K protein and m RNA in miR-199a-3p mimics cells decreased significantly, and the expression level of anti apoptotic protein Bcl-2 was significantly reduced and the expression level of autophagic protein decreased significantly in.3. three esophageal squamous cell carcinoma cells, compared with those in the untransfected group and the cells transfected with the negative control of the miR-199a-3p mimics. The expression level of /II protein was significantly increased in.4. three esophageal squamous cell carcinoma cells. Compared with the control group, there was no significant difference in the expression of miR-199a-3p in the ECa109 cells of.5. esophageal squamous cell carcinoma with RAD001. Compared with the untreated group, the number of cell clones decreased significantly after the Rictor down-regulation cells were treated with the PI3K/AKT/m TOR pathway inhibitor. The number of apoptotic cells increased significantly. Conclusion 1.miR-199a-3p can inhibit the proliferation of esophageal squamous cell carcinoma cells and induce apoptosis. The mechanism may enhance the effect of PI3K/AKT/m TOR signaling pathway inhibitor on cell proliferation and apoptosis after the downregulation of Rictor expression by M TORC1/p70S6K signaling pathway and autophagy related.2..
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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