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0.0012、<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�
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=0.0012,<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 express
作者
陈选才
符浩
唐亚纯
唐昕
祝乐喜
李雪峰
朱子贵
CHEN Xuancai;FU Hao;TANG Yachun;TANG Xin;ZHU Lexi;LI Xuefeng;ZHU Zigui(Department of Urology,Nanhua Hospital Affiliated to Hengyang Medical College of Nanhua University,Hengyang 421002,Hunan Province,China;不详)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2023年第12期1442-1449,共8页
Chinese Journal of Biologicals
基金
湖南省自然科学基金(2019JJ50550)
湖南省卫健委科研课题(B202304056903)。
