MiRNA-451调控人脑胶质瘤细胞上皮—间质转化现象的机制研究

发布时间：2018-03-11 05:34

本文选题：神经胶质瘤　切入点：miRNA-451　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究过表达mi RNA-451后调控人脑LN229和U251胶质瘤细胞系上皮-间质转化的机制,以及过表达mi RNA-451后对前述细胞株的生物学行为的影响(侵袭、迁移、增殖等)。方法:第一部分:寻找并验证mi RNA-451对人脑神经胶质瘤细胞系的上皮-间质转化作用及其影响路径。具体方案是:选取LN229和U251人脑胶质瘤细胞系进行培养,Lipofectamine 2000转染试剂进行转染构建高表达mi RNA-451细胞株。实验分为空白对照组不进行任何处理;无义序列组:转染寡核苷酸无意义序列为5-ACGUGACACGUUCGGAGAATT-3,5-UUCUCCGAACGUCACGUTT-3;和mi RNA-451处理组,转染mi RNA-451拟似物(mi RNA-451mimics)序列为5-AAACCGUACCAUUACUGAGUU-3,3-CUCAGUAAUGGUAACGGUUUU-5,(简称mi RNA-451);实时定量聚合酶链反应(RT-PCR)的研究能够对神经胶质瘤细胞系各组细胞中的转染后mi RNA-451的表达水平进行清晰准确的衡量,Western Blot实验验证E钙黏素(E-cadherin),N钙黏素(N-cadherin),波形蛋白(Vimentin),Twist,Snail的蛋白相对表达量;通过免疫荧光实验进而比较分析mi RNA-451处理组与空白对照组的N-cadherin、Vimentin的蛋白表达量;计算机蛋白交联网络软件以及前期研究成果构建mi RNA-451调控EMT发生相关蛋白的信号通路;Western Blot实验验证mi RNA-451处理组中mi RNA-451调控细胞EMT发生信号通路中相关目的蛋白(CAB39、AMPK、PI3K、AKT、磷酸化AKT)的相对蛋白表达量。第二部分:分析mi RNA-451对人脑神经神经胶质瘤细胞系生物行为的影响。按照第一部分的方法转染并分组,定量聚合酶链反应(RT-PCR)研究结果证明神经胶质瘤细胞系各组细胞中的mi RNA-451转染后的表达水平;Transwell实验对高表达mi RNA-451后抑制胶质瘤细胞株的侵袭能力做出证实;细胞划痕实验对高表达mi RNA-451后胶质瘤细胞株迁移能力做出有效检测;使用细胞计数盒(CCK-8)和单克隆实验验证高表达mi RNA-451后胶质瘤细胞株的增殖能力;Western blot实验能够对神经胶质瘤细胞株生物学行为相关目的蛋白表达量做出蛋白层面的验证。结果:第一部分结果显示:在和空白对照组和无义序列组的分析对比中,mi RNA-451处理组的该基因表达量有显著的增加;Western Blot表明mi RNA-451处理组中的E-cadherin表达量显著上升,Twist、Snail、N-cadherin、Vimentin的蛋白表达量显著减少;差异都具有统计学意义(P0.05);在免疫荧光实验中,所取得实验结果证实在mi RNA-451处理组中N-cadherin、Vimentin的蛋白表达量比空白对照组减少明显,且结果具有显著性(P0.05),与Western Blot实验发现相同;在具体的信号通路图中可以CAB39基因能够通过AMPK,PI3K/AKT通路对EMT相关蛋白的表达产生影响,进而对癌细胞的行为的产生作用,我们前期研究已经证实CAB39是mi RNA-451的靶标基因;Western blot实验结果证明mi RNA-451处理组mi RNA-451调控EMT相关蛋白信号通路中相关目的蛋白CAB39、AMPK、PI3K、磷酸化AKT等的表达,且实验结果发现表达量均减少了,并均有统计学意义(P0.05)。第二部分结果统计发现:与空白对照组和无义序列相比,RT-PCR实验有效说明了mi RNA-451处理组中其表达量的上升显著;Transwell实验有效说明了mi RNA-451处理组细胞侵袭能力的下降显著;细胞划痕实验对mi RNA-451处理组细胞迁移能力的抑制减弱结果做出了充分有效说明;细胞计数盒(CCK-8)和单克隆实验有效说明了mi RNA-451处理组的神经胶质瘤细胞殖能力被减弱;Western blot实验对mi RNA-451组神经胶质瘤细胞生物学行为相关的蛋白Cyclin D1、MMP-2、MMP-9的表达量的显著减少做出了有效充分说明,且实验统计结果显著性水平较高(P0.05)。结论:通过上述实验及实验结果统计分析,可以发现mi RNA-451靶向的CAB39能够通过AMPK、PI3K/AKT通路对胶质瘤细胞系中上皮-间质转化的发生产生较为显著的抑制作用;并在其基础上对神经胶质瘤细胞株的侵袭和迁移能力产生明显的限制;并且可以使其克隆增殖能力受限。
[Abstract]:Objective: To study the expression of MI RNA-451 and U251 LN229 after the regulation of human brain glioma cell lines of epithelial mesenchymal transition mechanism, and the effect of overexpression of MI RNA-451 on the cell biological behavior (invasion, migration, proliferation, etc.). Methods: the first part: to find and verify mi RNA-451 on human glioma tumor cells of epithelial mesenchymal transition and its influence path. Specific programs are: LN229 and U251 were cultured from human glioma cell line, Lipofectamine 2000 transfection reagent were transfected with MI expressing RNA-451 cells were divided into blank control group without any treatment; nonsense sequence group: nonsense oligonucleotide transfection sequence 5-ACGUGACACGUUCGGAGAATT-3,5-UUCUCCGAACGUCACGUTT-3; and MI RNA-451 treatment group, Mi transfection of RNA-451 analogues (MI RNA-451mimics) sequence for 5-AAACCGUACCAUUACUGAGUU-3,3-CUCAGUAAUGGUAACGG UUUU-5 (MI RNA-451); real time quantitative polymerase chain reaction (RT-PCR) to study the expression level of MI RNA-451 of transfected glioma cell line cells were measured in the clear, Western Blot experimental verification of E cadherin (E-cadherin), N cadherin (N-cadherin), vimentin (Vimentin, Twist), the relative expression of Snail protein by immunofluorescence; experimental and comparative analysis of MI RNA-451 treatment group and blank control group N-cadherin, the expression of Vimentin protein; computer network software and the previous research results to construct protein cross-linking signaling pathway mi RNA-451 regulation EMT related protein; MI RNA-451 regulate cell associated EMT the purpose of signal pathway of Western protein in Blot experiment mi RNA-451 treatment group (CAB39, AMPK, PI3K, AKT, phosphorylated AKT) expression of relative protein content. The second part: the analysis of MI RNA-451 Effect on the human brain glioma cell line biological behavior. Grouped according to the first part of the method of transfection and quantitative polymerase chain reaction (RT-PCR) results show that the expression level of MI RNA-451 was transfected into glioma cell line cells were in the post; the invasion ability of Transwell experiments on the high expression of MI inhibits glioma cell line RNA-451 make validation; cell scratch experiments on expression of MI after RNA-451 glioma cell migration ability to make effective use of detection; cell (CCK-8) and experimental verification of high expression of monoclonal proliferation of glioma cells line mi RNA-451 Western blot; experiments on biological behavior of glioma cell lines related to protein expression the amount of protein to test level. Results: the results showed that: in the first part and the blank control group and antisense sequence group comparative analysis, MI RNA-451 The expression of the gene group is significantly increased; Western Blot showed significantly increased expression of MI, RNA-451 group in E-cadherin Twist, Snail, N-cadherin, Vimentin protein expression was significantly reduced; all the differences were statistically significant (P0.05); in immunofluorescence experiments, the experimental results show that N-cadherin in MI RNA-451 treatment group, the expression of Vimentin protein decreased significantly than the control group, and the result was significant (P0.05), Western and Blot found the same experiment; in signaling pathway specific in CAB39 gene by AMPK, affect the expression of PI3K/AKT pathway related proteins of EMT, and then influences the behavior of the cancer cells, we demonstrated that CAB39 is the target gene mi RNA-451 Western blot; the experimental results show that the MI RNA-451 mi RNA-451 EMT control group related protein signal pathway In the related protein CAB39, AMPK, PI3K, the expression of phosphorylated AKT, and the experimental results show that the expression amount was reduced, and had statistical significance (P0.05). Some results of second statistics: compared with the blank control group and antisense sequence, RT-PCR experiment shows mi RNA-451 treatment group in the expression the Transwell increased significantly; experimental effective description of the MI RNA-451 group cell invasion ability decreased significantly; the inhibition of cell scratch experiment on MI RNA-451 treated cell migration results adequately weakened effective instruction; cell count Kit (CCK-8) and the effective description of the MI monoclonal RNA-451 treated glioma cell proliferation the ability of MMP-2 Western blot has been weakened; experimental group RNA-451 on MI glioma cell biological behavior of protein Cyclin, D1 significantly reduced the expression of MMP-9 has made the full said In Ming Dynasty, and the experimental results were significantly higher (P0.05). Conclusion: through the analysis of the experiment and the experimental results of statistics, can be found in CAB39 Mi to target RNA-451 through AMPK, PI3K/AKT pathway on epithelial glioma cell lines between matter transformation inhibited more significantly; and on the basis of on the migration and invasion of glioma cell lines have been limited; and the clonal proliferation ability is limited.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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