MiR-483-3P下调RIOK3蛋白表达促进神经母细胞瘤的增殖和迁移

摘要

Objetive To explore the influence of microRNA—483 —3P (miR—483 —3P)on neuroblastoma cells'proliferation,invasion and migration abilities,and predict target genes for miR —483 —3P,and also to dis-cuss its impact on the target gene. Methods miRNA array results showed that miR—483 —3P ,which has the most significant difference in the changed miRNAS,was upregulated in neuroblastoma.Under the use of cationic liposomes LipofectamineTM2000 ,chemically synthesized miR—483 —3P inhibitor or a negative control sequence of miR—483 —3P inhibitor were transiently transfected into human neuroblastoma SH—SY—5Y cell lines.The ex-pression of miR—483 —3P was detected by using quantitative Real-time PCR technique.The proliferation and mi-gration of neuroblastoma cells were examined in vitro by CCK—8 and transwell experiment assay.The bioinfor-matics software was used to predict target genes of miR—483 —3P and luciferase reporter gene detection experi-ments,Western blot experiments were proformed to validate the target gene. Results Compared with adjacent none-tumor tissues,miR—483 —3P was upregulated in neuroblastoma tissues(P <0.01).Compared with nega-tive control,the miR—483 —3P expression was decreased after miRNA inhibitor transfection(P <0.01),the pro-liferation and migration of cells in vitro are all decreased(P <0.05).Bioinformatics software predicted RIOK3 is one of the target genes of miR—483 —3P,when cells transfected with miR—483 —3P inhibitor the luciferase ac-tivity would increased(P <0.01).Compared with the negative control group,when cells transfected with miR—483 —3P inhibitor at the protein level RIOK3 would increased(P <0.01). Conclusion RIOK3 is one of the target genes of miR—483 —3P,also miR—483 —3P can down regulate the expression of RIOK3 and promote the proliferation and migration abilities of neuroblastoma cell.%目的：探讨 microRNA —483—3P（miR—483—3P）对神经母细胞瘤细胞的增殖、侵袭及迁移能力的影响，预测并验证 miR—483—3P 的靶基因及其对靶基因的影响。方法miRNA 微阵列芯片结果发现 miR—483—3P 在神经母细胞瘤中表达上调，差异倍数显著，并居于差异表达 miRNA 的首位。利用阳离子脂质体 LipofectamineTM2000将化学合成的 miR—483—3P inhibitor、miR—483—3P inhibitor 的阴性对照序列分别瞬时转染入人神经母细胞瘤 SH—SY —5Y 细胞株中，RT—qPCR 技术检测各组中 miR—483—3P 的表达水平，CCK—8法检测各组癌细胞的增殖情况，Transwell 小室实验检测各组癌细胞的体外侵袭和迁移能力。利用生物信息学软件预测 miR—483—3P 的靶基因并用荧光素酶报告基因检测实验、West-ern blot 实验加以验证。结果与癌旁正常组织相比，miR —483—3P 在神经母细胞瘤中高表达（P ＜0.01）；与阴性对照组相比，癌细胞转染 miR —483—3P inhibitor 后，miR —483—3P 表达显著下调（P ＜0.01），细胞的增殖情况和体外迁移能力均降低（P ＜0.05）。生物信息学软件预测出 RIOK3是 miR—483—3P 的靶基因之一，当细胞转染了 miR—483—3P inhibitor 后荧光酶活性升高（P ＜0.01）。与阴性对照组相比，当细胞转染了 miR —483—3P inhibitor 后在蛋白质水平 RIOK3表达升高（P ＜0.01）。结论RIOK3是 miR—483—3P 的靶基因之一，miR—483—3P 能下调 RIOK3的表达并显著促进神经母细胞瘤细胞的增殖和体外迁移。