摘要目的:探讨肿瘤抑制因子微小RNA(miRNA，miR)-149-5p对肾癌细胞转移潜能的影响及机制。方法:GRC-1细胞分成miR-149-5p组(转染miR-149-5p mimics)、miR-NC组(转染mimics control)、miR-149-5p＋丝氨酸-精氨酸蛋白激酶1(SRPK1)组(共转染miR-149-5p mimics、pcDNA3.1-SRPK1)、miR-149-5p＋vector组(共转染miR-149-5p mimics、pcDNA3.1)。实时定量反转录聚合酶链反应(RT-qPCR)检测miR-149-5p水平，Transwell小室测定细胞侵袭和迁移能力变化，蛋白质印迹法(Western blot)检测细胞中N-钙黏蛋白(N-cadherin)、E-钙黏蛋白(E-cadherin)蛋白水平。利用在线靶基因预测软件发现SRPK1可能是miR-149-5p的靶基因，双荧光素酶报告系统鉴定靶向关系。应用SPSS 21.0统计软件分析。结果:miR-149-5p组的GRC-1细胞中miR-149-5p(2.69±0.27比1.01±0.08)和E-cadherin(0.81±0.09比0.42±0.05)水平显著高于miR-NC组，侵袭数目[(70.81±6.35)个比(110.64±9.67)个] 、迁移数目[(108.46±9.27)个比(162.76±12.71)个]、N-cadherin(0.46±0.05比0.79±0.11)和SRPK1(0.45±0.06比0.92±0.08)蛋白水平显著低于miR-NC组，差异均有统计学意义( FmiR-149-5p＝95.385， F侵袭数目＝20.216， F迁移数目＝22.010， FN-cadherin＝15.100， FE-cadherin＝31.331， FSRPK1＝31.785， P<0.05)。共转染SRPK1野生型质粒的miR-149-5p组细胞荧光素酶活性(0.37±0.04比1.00±0.11)显著低于miR-NC组，差异有统计学意义( t＝16.150， P<0.05)。共转染SRPK1突变型质粒的miR-149-5p组细胞荧光素酶活性(0.99±0.10比1.00±0.09)与miR-NC组比较差异无统计学意义( t＝0.220， P>0.05)。miR-149-5p＋SRPK1组的GRC-1细胞中E-cadherin(0.45±0.04比0.80±0.08)水平显著低于miR-149-5p＋vector组，侵袭数目[(99.51±7.48)个比(71.84±5.37)个]、迁移数目[(145.06±11.14)个比(107.63±10.20)个]、N-cadherin(0.86±0.10比0.47±0.04)和SRPK1(0.89±0.06比0.50±0.07)蛋白水平显著高于miR-149-5p＋vector组，差异均有统计学意义( tSRPK1＝7.327， t侵袭数目＝5.205， t迁移数目＝4.292，tN-cadherin＝6.272， tE-cadherin＝6.778， P<0.05)。 结论:肿瘤抑制因子miR-149-5p靶向负调控SRPK1表达降低肾癌细胞的迁移和侵袭能力。

abstractsObjective:To elucidate the role and mechanism of tumor suppressor microRN (miRNA, miR)-149-5p in metastasis potentials of renal cell carcinoma.Methods:GRC-1 cells were divided into miR-149-5p group(transfected with miR-149-5p mimics), miR-NC group(transfected with mimics control), miR-149-5p+ SRPK1 group(co transfected with miR-149-5p mimics and pcDNA3.1-SRPK1), miR-149-5p+ vector group(co transfected with miR-149-5p mimics and pcDNA3.1). The transfection efficiency of miR-149-5p was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The cell invasion and migration ability were measured by Transwell chamber. The levels of N-cadherin and E-cadherin proteins were detected by Western blotting. SRPK1 may be a target gene of miR-149-5p by using online target gene prediction software. Targeting relationship was identified by double luciferase reporting system.Results:The levels of miR-149-5p (2.69±0.27 vs. 1.01±0.08) and E-cadherin (0.81±0.09 vs. 0.42±0.05)in the miR-149-5p group were significantly higher than those in the miR-NC group, and invasion number [(70.81±6.35) vs. (110.64±9.67)], migration number [(108.46±9.27)vs. (162.76±12.71)], N-cadherin (0.46±0.05 vs. 0.79±0.11)and SRPK1 (0.45±0.06 vs. 0.92±0.08)proteins levels were significantly lower than miR-NC group, and the difference was statistically significant ( FmiR-149-5p=95.385, Finvasion number=20.216, Fmigration number=22.010, FN-cadherin=15.100, FE-cadherin=31.331, FSRPK1=31.785, P<0.05). The luciferase activity of the miR-149-5p group (0.37±0.04 vs. 1.00±0.11) co-transfected with the SRPK1 wild type plasmid was significantly lower than that of the miR-NC group, the difference was statistically significant ( t=16.150, P<0.05). The luciferase activity of the miR-149-5p group (0.99±0.10 vs. 1.00±0.09) co-transfected with the SRPK1 mutant plasmid was not significantly different from that of the miR-NC group ( t=0.220, P>0.05). The level of E-cadherin (0.45±0.04 vs. 0.80±0.08) in the miR-149-5p+ SRPK1 group was significantly lower than that in the miR-149-5p+ vector group, and the invasive number [(99.51±7.48) vs. (71.84±5.37)], migration number [(145.06±11.14) vs. (107.63±10.20)], N-cadherin (0.86±0.10 vs. 0.47±0.04) and SRPK1 (0.89±0.06 vs. 0.50±0.07) proteins levels were significantly higher than the miR-149-5p+ vector group, and the difference was statistically significant ( tSRPK1=7.327, tinvasion number=5.205, tmigration number=4.292, tN-cadherin=6.272, tE-cadherin=6.778, P<0.05). Conclusion:Tumor suppressor miR-149-5p targeting negative regulation of SRPK1 expression reduces the migration and invasion of renal cancer cells.