MiR-483-3P下调RIOK3蛋白表达促进神经母细胞瘤的增殖和迁移

MiR-483-3P down regulate RIOK3 expression and promote the proliferation and migration ability of human neuroblastoma cells

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人人神经母细胞瘤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、Western 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。

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 migration of neuroblastoma cells were examined in vitro by CCK-8 and transwell experiment assay. The bioinformatics software was used to predict target genes of miR-483-3P and luciferase reporter gene detection experiments, 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 transfeetion（ P 〈 0. 01 ） , the proliferation and migration of cells in vitro are all decreased（P 〈 0. 05 ）. Bioinformaties software predicted RIOK3 is one of the target genes of miR-483-3P ,when cells transfected with miR-483-3P inhibitor the lueiferase activity 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.