【摘要】 目的 探讨miR-130b-5p靶向E26转录因子1(E26 transformation specific-1,ETS1)对前列腺癌（prostatic cancer,PCa）细胞增殖、迁移及侵袭的影响及其作用机制。方法 qRT-PCR法测定PCa组织及其癌旁组织、PCa细胞（LNCap、PC-3、DU-145）及正常前列腺细胞（RPWE-1）中miR-130b-5p基因mRNA的转录水平，Western blot法检测PCa细胞中ETS1蛋白的表达水平。生物信息学、双荧光素酶活性测定法、qRT-PCR法及Western blot法预测和验证miR-130b-5p与ETS1的靶向关系。将PC-3细胞分为对照（不作任何处理）、mimic（转染miR-130b-5p模拟物）、mimic+ETS1组（转染miR-130b-5p模拟物+pcDNA-ETS1），采用克隆形成试验和CCK-8法分别检测细胞增殖情况及活力，划痕试验和Transwell小室试验分别检测细胞迁移及侵袭情况，Western blot法检测侵袭相关蛋白及PI3K/AKT/mTOR通路相关蛋白的表达水平。结果 与癌旁组织比较，PCa组织中miR-130b-5p基因mRNA的转录水平明显下降（t=12.450,P <0.001）；与RPWE-1细胞比较，LNCap、PC-3和DU-145细胞中miR-130b-5p基因mRNA的转录水平均明显下降（t分别为4.463、7.103和5.741,P分别为0.001 2、<0.001和<0.001）,ETS1蛋白表达水平显著升高（t分别为4.850、9.325和7.723,P分别为0.008、<0.001和0.002）。miR-130-5p可以靶向负调控ETS1的表达。与对照组比较，mimic组细胞克隆率、细胞活力、细胞划痕愈合率均明显降低（t分别为11.370、10.640、15.660,P均<0.001），侵袭细胞数明显减少（t=10.160,P <0.001），基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、MMP-9和波形蛋白（vimentin）表达水平显著下调（t分别为15.120、9.992和12.600,P分别为<0.001、<0.001和0.002），钙黏蛋白（E-cadherin）表达显著升高（t=6.928,P <0.001），磷脂酰肌醇-3-激酶（phosphatidylinositol-3 kinase,PI3K）、蛋白激酶B(protein kinase B,AKT)和哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）磷酸化水平均显著降低（t分别为7.746、8.041和11.510,P分别为0.002、0.002、<0.001）；与mimic组比较，mimic+ETS1组细胞克隆率、细胞活力、细胞划痕愈合率均明显升高（t分别为6.988、6.642、6.660,P均<0.001），侵袭细胞数明显增多（t=4.082,P=0.002）,MMP-2、MMP-9和vimentin蛋白表达水平显著上调（t分别为10.410、6.754和8.521,P分别为0.002、0.003、0.002）,E-cadherin表达水平显著下调（t=4.648,P <0.01）,PI3K、AKT和mTOR磷酸化水平均显著升高（t分别为4.850、4.323和10.840,P分别为0.008、0.008和<0.001）。结论 miR-130b-5p可靶向ETS1抑制PC-3细胞的增殖、迁移及侵袭，可能是通过调控PI3K/AKT/mTOR信号通路实现的。更多还原

【Abstract】 Objective To investigate the effects of miR-130b-5p targeting E26 transformation specific-1(ETS1)on proliferation,migration and invasion of prostatic cancer(PCa)cells and its mechanism. Methods The mRNA transcription level of miR-130b-5p gene in PCa tissues,adjacent tissues,(LNCap,PC-3,DU-145)and normal prostate cells(RPWE-1)PCa cells was measured by qRT-PCR,and the expression of ETS1 protein in PCa cells was detected by Western blot. Bioinformatics,fluorescein experiment,qRT-PCR and Western blot were used to predict and verify the targeting relationship between miR-130b-5p and ETS1. PC-3 cells were divided into control group(without any treatment),mimic group(transfected with miR-130b-5p mimic)and mimic + ETS1 group(transfected with miR-130b-5p mimic + pcDNA-ETS1). The cells were detected for the proliferation and viability by clone formation assay and CCK-8 respectively,measured for the migration and invasion by scratch test and Transwell chamber assay,and detected for the expression of invasion-related proteins and PI3K/AKT/mTOR pathway-related proteins by Western blot. Results The transcription level of miR-130b-5p mRNA in PCa tissues was significantly lower than that in adjacent tissues(t = 12. 450,P < 0. 001);Compared with RPWE-1 cells,the transcription level of miR-130b-5p mRNA in LNCap,PC-3 and DU-145 cells decreased significantly(t = 4. 463,7. 103 and 5. 741,P = 0. 001 2,< 0. 001 and < 0. 001,respectively),while the expression level of ETS1protein increased significantly(t = 4. 850,9. 325 and 7. 723,P = 0. 008,< 0. 001 and = 0. 002,respectively). miR-130-5p targeted and negatively regulated the expression of ETS1. Compared with the control group,the cloning rate,viability and scratch healing rate of cells in mimic group decreased significantly(t = 11. 370,10. 640 and 15. 660,respectively,each P < 0. 001),the number of invasive cells decreased significantly(t = 10. 160,P < 0. 001),the expression levels of matrix metalloproteinase-2(MMP-2),MMP-9 and vimentin decreased significantly(t = 15. 120,9. 992 and 12. 600,P < 0. 001,< 0. 001 and = 0. 002,respectively),while the expression level of E-cadherin increased significantly(t = 6. 928,P < 0. 001),and the phosphorylation levels of phosphatidylinositol-3 kinase(PI3K),protein kinase B(AKT)and mammalian target of rapamycin(mTOR)decreased significantly(t = 7. 746,8. 041 and 11. 510,P = 0. 002,0. 002,and < 0. 001,respectively);Compared with mimic group,the cell cloning rate,viability,scratch healing rate significantly increased in mimic + ETS1 group(t = 6. 988,6. 642 and 6. 660,respectively,each P < 0. 001),the number of invasive cells significantly increased(t = 4. 082,P = 0. 002),the expression levels of MMP-2,MMP-9 and vimentin proteins were significantly up-regulated(t = 10. 410,6. 754 and 8. 521,P = 0. 002,0. 003 and 0. 002,respectively),however,the expression level of E-cadherin was significantly down-regulated(t = 4. 648,P < 0. 01),and the phosphorylation levels of PI3K,AKT and mTOR were significantly up-regulated(t = 4. 850,4. 323 and 10. 840,P = 0. 008,0. 008 and < 0. 001,respectively)Conclusion miR-130b-5p targets ETS1 to inhibit the proliferation,migration and invasion of PC-3 cells,which may be through the regulation of PI3K/AKT/mTOR signaling pathway.更多还原