miR-483-3P下调RIOK3蛋白表达并促进神经母细胞瘤的增殖侵袭和迁移

发布时间：2017-12-31 08:36

本文关键词：miR-483-3P下调RIOK3蛋白表达并促进神经母细胞瘤的增殖侵袭和迁移　出处：《青岛大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的通过实验研究micro RNA-483-3P(mi R-483-3P)对神经母细胞瘤细胞的生物学行为的影响,诸如增殖能力、侵袭能力及迁移能力。利用生物信息学软件预测mi R-483-3P的靶基因并加以验证,以及探索mi R-483-3P对所预测的靶基因的影响。方法mi RNA微阵列芯片(Micro Array)的结果发现,与原发瘤相比,mi R-483-3P在神经母细胞转移瘤中表达水平明显上调,差异倍数显著,并居于所检测出的差异表达的mi RNA的第一位。利用阳离子脂质体Lipofectamine TM2000将化学合成的mi R-483-3P inhibitor、mi R-483-3P inhibitor的阴性对照序列(negative control)分别转染入人神经母细胞瘤SH-SY-5Y细胞株中,实时荧光定量聚合酶链式反应(RT-q PCR)技术检测各组神经母细胞瘤经处理后mi R-483-3P的表达水平,细胞计数试剂盒(CCK-8)法检测各组神经母细胞瘤细胞的增殖情况,Transwell小室实验检测各组神经母细胞瘤细胞的体外侵袭以及迁移能力。利用多个靶基因预测数据库,综合预测mi R-483-3P的靶基因,荧光素酶报告基因检测实验、Western blot实验加以验证。结果实验所取的9对神经母细胞瘤组织与癌旁正常组织,RT-QPCR结果显示,mi R-483-3P在神经母细胞瘤肿瘤组织中表达水平明显高于癌旁正常组织(P0.01);与转染了mi R-483-3P inhibitor的阴性对照组相比,癌细胞转染mi R-483-3P inhibitor后,mi R-483-3P被抑制表达水平显著下调(P0.01),细胞的增殖情况和体外迁移能力均降低(P0.05)。经多个靶基因生物信息学软件分析,预测出RIOK3是mi R-483-3P的靶基因之一,当神经母细胞瘤细胞转染了mi R-483-3P inhibitor后荧光酶的活性明显升高(P0.01)。与mi R-483-3P inhibitor阴性对照组相比,当细胞转染了mi R-483-3P inhibitor后,RIOK3蛋白的表达水平明显升高(P0.01)。结论RIOK3是miR-483-3P的靶基因之一,miR-483-3P能下调RIOK3蛋白的表达,mi R-483-3P可显著促进神经母细胞瘤细胞的增殖及体外迁移能力。
[Abstract]:Objective to investigate the effects of micro RNA-483-3P(mi R-483-3P on the biological behavior of neuroblastoma cells, such as proliferation. Invasive ability and migration ability. The target gene of mi R-483-3P was predicted and verified by bioinformatics software. And to explore the effect of mi R-483-3P on the predicted target gene. Methods the results of mi RNA microarray microarray microarray showed that compared with the primary tumor. The expression of mi R-483-3P was up-regulated in neuroblastoma, and the difference was significant. The chemically synthesized mi R-483-3P was chemically synthesized using cationic liposome Lipofectamine TM2000. Inhibitor. Negative control sequence of mi R-483-3P inhibitor. The cells were transfected into human neuroblastoma SH-SY-5Y cell lines. Real-time quantitative polymerase chain reaction (RT-q PCR) technique was used to detect the expression of mi R-483-3P in neuroblastoma after treatment. The proliferation of neuroblastoma cells in each group was detected by CCK-8 assay. The invasiveness and migration ability of neuroblastoma cells in each group were detected by Transwell chamber assay. The target genes of mi R-483-3P were comprehensively predicted by using multiple target gene prediction databases. Results 9 pairs of neuroblastoma tissues and adjacent normal tissues were detected by RT-QPCR. The expression of miR-483-3P in neuroblastoma was significantly higher than that in adjacent normal tissue (P 0.01). Compared with the negative control group transfected with mi R-483-3P inhibitor, the cancer cells were transfected with mi R-483-3P inhibitor. Mi R-483-3P was significantly down-regulated (P0.01). The cell proliferation and migration ability in vitro were decreased (P 0.05). It was predicted that RIOK3 was one of the target genes of mi R-483-3P by multiple target gene bioinformatics software analysis. The activity of fluorescence enzyme increased significantly after transfection of mi R-483-3P inhibitor into neuroblastoma cells (P 0.01). Compared with mi R-483-3P inhibitor negative control group. When the cells were transfected with mi R-483-3P inhibitor. Conclusion RIOK3 is one of the target genes of miR-483-3P. MiR-483-3P can down-regulate the expression of RIOK3 protein. Mi R-483-3P can significantly promote the proliferation and migration of neuroblastoma cells in vitro.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.4

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