摘要
目的研究miR-145靶向调控锌指蛋白转录因子5(KLF5)抑制膀胱癌BIU-87细胞增殖、侵袭、迁移的机制。方法miR-145 mimics对BIU-87细胞进行转染,实验分为对照组、miR-145阴性对照组、miR-145 mimics组,SV-HUC-1细胞作为SV-HUC-1组。实时荧光定量法(RT-qPCR)测定转染后BIU-87细胞中miR-145表达水平,蛋白印迹法(WB)测定KLF5、PI3K、AKT、p-AKT、PCNA、MMP-9、MMP-2、E-cadherin表达水平,MTT法测定转染后BIU-87细胞活性,Transwell小室实验测定转染后BIU-87细胞侵袭能力,划痕实验测定转染后BIU-87细胞体外迁移能力。TargetScan数据库预测miR-145的靶基因并采用荧光素酶报告实验进行验证。结果与SV-HUC-1细胞组相比,BIU-87细胞对照组miR-145表达水平明显降低(P<0.05),KLF5、PI3K表达水平和AKT磷酸化水平显著升高(P<0.05);与对照组、miR-145阴性对照组相比,miR-145 mimic组BIU-87细胞活性、侵袭能力、迁移能力明显降低(P<0.05),miR-145、E-cadherin表达水平显著升高(P<0.05),KLF5、PI3K、PCNA、MMP-9、MMP-2表达水平和AKT磷酸化水平明显降低(P<0.05)。TargetScan数据库预测显示KLF5是miR-145靶基因,双荧光素酶实验证实KLF5是miR-145作用靶点。结论miR-145靶向下调KLF5表达水平,阻断PI3K/AKT通路激活从而抑制膀胱癌BIU-87细胞增殖、侵袭、迁移。
Objective To explorethe mechanism of miR-145 targeting and regulating zinc finger protein transcription factor 5(KLF5)to inhibit the proliferation,invasion and migration of bladder cancer BIU-87 cells.Methods BIU-87 cells were transfected with miR-145 mimicsand divided into control group,miR-145 negative control group and miR-145 mimics group,SV-HUC-1 cells were as SV-HUC-1 cell group.Real time fluorescence quantitative method(RT qPCR)was used to detect the expression of miR-145 in BIU-87 cells.The expression levels of KLF5,PI3K,AKT,p-AKT,PCNA,MMP-9,MMP-2 and E-cadherin were measured by Western blot.The activity of BIU-87 cells was measured by MTT.Andthe invasiveness of BIU-87 cells was measured by Transwell cell assay.The migration ability of BIU-87 cells in vitro was measured by scratch test.The target gene of miR-145 was predicted by TargetScandatabase and verified by Luciferase Report experiment.ResultsCompared with SV-HUC-1 cell group,the expression level of miR-145 in BIU-87 cell control group decreased significantly(P<0.05).The expression levels of KLF5,PI3K and phosphorylation level of AKT increased significantly(P<0.05).Compared with the control group and miR-145 negative control group,the activity,invasion and migration of BIU-87 cells in miR-145 mimic group were significantly decreased(P<0.05).The expression levels of miR-145 and E-cadherin increased significantly(P<0.05).And the expression levels of KLF5,PI3K,PCNA,MMP-9,MMP-2 and phosphorylation level of AKT decreased significantly(P<0.05).The prediction was performedby TargetScandatabase which showed that KLF5 was the target gene of miR-145.And KLF5 was the target of miR-145 confirmed by the experimentof double luciferase.Conclusions MiR-145 can down regulate the expression of KLF5 and block the activation of PI3K/Akt pathway so as to inhibit the proliferation,invasion and migration of BIU-87 cells.
作者
孔令伟
吕宪宝
KONG Lingwei;LYU Xianbao(Department of Urology, Chengwu Hospital Affiliated to Taishan Medical College & Chengwu County People's Hospital, Tai'an 274200,China)
出处
《中国肿瘤外科杂志》
CAS
2021年第1期70-75,85,共7页
Chinese Journal of Surgical Oncology
关键词
MIR-145
锌指蛋白转录因子
增殖
侵袭
迁移
miR-145
Zinc finger protein transcription factor
Proliferation
Invasion
Migration
