肾细胞癌组织中高表达的miRNA let-7i-5p通过透明质酸结合蛋白4调控769-P细胞的恶性生物学行为

Highly expressed miRNA let-7i-5p in renal cell carcinoma tissues regulates the malignant biological behaviors of 769-P cells through hyaluronan-binding protein 4

目的:探讨miRNA let-7i-5p在肾细胞癌(renal cell carcinoma,RCC)组织中的表达水平及其对RCC细胞769-P的增殖、迁移、侵袭和透明质酸结合蛋白4(hyaluronan-binding protein 4,HABP4)表达的影响。方法:利用TCGA RCC数据库及GEO数据库对let-7i-5p在RCC组织中的表达进行meta分析。体外常规培养人RCC细胞769-P并对其进行转染,根据转染物不同分为过表达组(转染let-7i-5p模拟物)、抑制组(转染let-7i-5p抑制物)和对照组(转染NC序列)。采用CCK-8、细胞划痕实验、Transwell实验及WB法检测let-7i-5p对769-P细胞增殖活力、划痕愈合率、穿膜侵袭细胞数及HABP4表达水平的影响。双荧光素酶报告基因实验验证let-7i-5p与HABP4 mRNA的靶向关系。结果:分析TCGA RCC数据库及5个GEO数据集(GSE23085、GSE47582、GSE95385、GSE16441和GSE71302)中数据结果显示,let-7i-5p在RCC组织中的表达水平显著高于正常肾组织(均P<0.05)。体外实验结果显示,与对照组相比,过表达组在24、48及72 h时细胞增殖活力显著升高,而抑制组显著降低(均P<0.01);过表达组的划痕愈合率[(37.276±2.058)%vs(15.663±2.949)%,P<0.01]和穿膜侵袭细胞数[(377.000±34.044)vs(255.667±25.384)个,P<0.01]均显著升高,而抑制组的划痕愈合率[(8.791±2.568)%vs(15.663±2.949)%,P<0.05]和穿膜侵袭细胞数[(170.333±14.978)vs(255.667±25.384)个,P<0.01]均显著降低。在野生型HABP4-3’URT质粒组中,过表达let-7i-5p可显著抑制细胞的荧光素酶活性(P<0.01);而在突变型HABP4-3’URT质粒组中,过表达let-7i-5p对细胞的荧光素酶活性无影响(NS,P>0.05)。WB检测结果显示,与对照组相比,过表达组的HABP4和E-cadherin的水平均降低(均P<0.01)、CDK2的水平升高(P<0.01),而抑制组则相反(均P<0.01)。结论:Let-7i-5p在RCC组织中呈高表达,其可能通过靶向HABP4基因来促进769-P细胞的增殖、迁移和侵袭。

Objective:To investigate the expression level of miRNA let-7i-5p in renal cell carcinoma(RCC)tissues and to explore its effect on the proliferation,migration,invasion,and hyaluronan-binding protein 4(HABP4)expression in human RCC 769-P cells.Methods:A meta-analysis of let-7i-5p expression in RCC tissues was performed using the TCGA RCC database and GEO database.The human RCC 769-P cells were routinely cultured for transfection in vitro,and were divided into three groups according to different transfection materials:overexpression group(transfected with let-7i-5p mimics),inhibition group(transfected with let-7i-5p inhibitors),and control group(transfected with NC sequence).CCK-8,cell scratch test,Transwell assay,and WB method were used to detect the effects of let-7i-5p on the proliferation,scratch healing rate,invaded cell numbers and HABP4 expression in 769-P cells.Dual[1]luciferase reporter gene assay was performed to demonstrate the targeting relationship between let-7i-5p and HABP4.Results:Data analysis of TCGA RCC database and 5 GEO data sets(GSE23085,GSE47582,GSE95385,GSE16441,and GSE71302)showed that the expression level of let-7i-5p was significantly higher in RCC tissues than in normal kidney tissues(all P<0.05).As shown by in vitro experiments,compared with the control group,the cell proliferation activity of the overexpression group was significantly increased at 24,48,and 72 h,while that of the inhibition group was decreased(all P<0.01).The scratch healing rate[(37.276±2.058)%vs(15.663±2.949)%,P<0.01]and the invaded cell numbers[(377.000±34.044)vs(255.667±25.34),P<0.05]were significantly increased in the overexpression group,while the scratch healing rate[(8.791±2.568)%vs(15.663±2.949)%,P<0.05]and the invaded cell numbers[(170.333±14.978)vs(255.667±25.384),P<0.01]were significantly reduced in the inhibition group.The luciferase activity was significantly inhibited by the overexpression of let-7i-5p in the wild-type HABP4-3'URT plasmid group(P<0.01),while no statistical difference was observed in the mutant HABP4-3'URT plasmid group(P>0.05).WB results showed that compared with the control group,the expression of HABP4 and E-cadherin in the overexpression group was decreased(all P<0.01),and the expression of CDK2 was increased(P<0.01),while the inhibition group showed the opposite trend(all P<0.01).Conclusion:Let-7i-5p is highly expressed in RCC tissues and promotes the proliferation,migration,and invasion of 769-P cells possibly by targeting HABP4.