MicroRNA-145通过ADAM28抑制人肾癌细胞A-498上皮-间充质转化功能

目的:探讨微小RNA(miR-145)对人肾癌细胞A-498上皮-间充质转化(EMT)功能的影响及其相关机制。方法:将A-498肾癌细胞株分别转染miR-145模拟物(M145)和模拟物阴性对照(MNC),分别作为M145组和MNC组,并设立空白对照(MC)组,采用RT-qPCR法检测各组细胞miR-145水平。Transwell实验检测3组细胞侵袭能力的变化。Western blot法检测3组细胞波型蛋白(vimentin)、E-cadherin和ADAM28表达水平。应用生物信息学方法预测miR-145的靶基因。采用Western blot法检测ADAM28过表达对miR-145抑制EMT的拮抗作用。双萤光素酶报告基因实验验证miR-145与ADAM28的关系。结果:与MC组相比,M145组miR-145的表达水平显著上调(P<0.05)。M145组侵袭细胞数量显著低于MC组(P<0.05)。M145组细胞vimentin蛋白表达量显著降低(P<0.05),E-cadherin蛋白表达量显著升高(P<0.05),ADAM28蛋白表达量显著降低(P<0.05)。ADAM28过表达M145组肾癌细胞中vimentin蛋白表达量显著升高(P<0.05),E-cadherin蛋白表达量显著降低(P<0.05)。双萤光素酶报告基因实验结果显示ADAM28为miR-145的下游靶基因。结论:miR-145可能通过降低下游靶基因ADAM28水平影响EMT相关蛋白表达,从而抑制人肾癌细胞A-498的EMT过程。

MicroRNA-145 inhibits epithelial-mesenchymal transition in human renal cancer A-498 cells by ADAM28

AIM:To investigate the inhibitory effect of microRNA-145(miR-145)on epithelial-mesenchymal transition(EMT)in renal cancer A-498 cells.METHODS:The A-498 cells were transfected with miR-145 mimics(M145)and mimic negative control(MNC),which served as M145 group and MNC group,respectively.Mock control(MC)group was set up using untreated A-498 cells.The expression level of miR-145 in each group was detected by RT-qPCR.Transwell assay was used to detect the invasion ability of the cells.The protein expression of vimentin,E-cadherin and ADAM28 was determined by Western blot.Bioinformatic method was used to predict the target genes of miR-145.Antagonistic effect of ADAM28 over-expression on the inhibition of EMT by miR-145 was detected by Western blot.The relationship between miR-145 and ADAM28 was analyzed by dual-luciferase reporter assay.RESULTS:The expression level of miR-145 in M145 group was significantly up-regulated than that in MC group(P<0.05).The number of invasive cells in M145 group was 12.78±3.37,which was significantly lower than that in MC group(P<0.05).ADAM28 may be the target gene of miR-145.Compared with MC group,the protein expression of vimentin and ADAM28 in M145 group was significantly decreased(P<0.05),while the protein expression of E-cadherin was significantly increased(P<0.05).After ADAM28 over-expression,the protein expression of vimentin in the A-498 cells of M145 group was significantly increased(P<0.05),and the protein expression of E-cadherin was significantly decreased(P<0.05).The results of dual-lucife-rase reporter assay showed that ADAM28 was a downstream target gene of miR-145.CONCLUSION:miR-145 may inhibit the expression of EMT-related proteins through the downstream target gene ADAM28 and inhibit the EMT process of renal cancer A-498 cells.