miR-708-5p通过靶向URGCP抑制非小细胞肺癌A549细胞侵袭、转移的研究

MiR-708-5p Inhibits Proliferation,Migration and Invasion of Non-Small Cell Lung Cancer A549 Cells by Targeting URGCP

目的探讨miR-708-5p对非小细胞肺癌A549细胞的增殖、迁移和侵袭的影响及其与上调基因-4(upregulated gene-4/upregulator of cell proliferation,URG4/URGCP)的靶向关系。方法选取非小细胞肺癌细胞株A549,正常肺成纤维细胞HLF-1为研究对象,根据A549细胞转染物质的不同,分为Control组(未进行任何处理),NC组(转染随机序列RNA Oligo),miR-708-5p组(转染miR-708-5p mimics)。MTT检测增殖能力,流式细胞术检测细胞周期,Transwell小室实验检测侵袭能力,划痕实验检测迁移能力,Western blot检测URGCP表达水平;实时荧光定量PCR检测miR-708-5p、URGCP基因mRNA表达水平;荧光素酶实验验证miR-708-5p与URGCP的靶向关系。结果与HLF-1细胞株相比,肺癌细胞株A549细胞miR-708-5p表达水平降低,URGCP-mRNA表达升高(P<0.05),同时,URGCP蛋白水平升高。转染72h后,与Control、NC组比较,miR-708-5p组细胞URGCP基因mRNA明显降低,URGCP蛋白表达量明显降低(P均<0.01)。miR-708-5p组细胞增殖、迁移和侵袭能力明显低于Control、NC组(P<0.05)。与WT-URGCP组相比,miR-708-5p+WT-URGCP组细胞荧光素酶活性明显降低(P<0.01)。结论miR-708-5p在非小细胞肺癌A549细胞中低表达,过表达miR-708-5p可抑制A549细胞增殖、迁移和侵袭,机制可能与负性调控URGCP有关。

Objective To investigate the effect of miR-708-5p on the proliferation,migration and invasion of non-small cell lung cancer A549 cells and its targeting relationship with up-regulated gene-4/up regulator of cell proliferation(URG4/URGCP).Methods Non-small cell lung cancer cell lines A549 and normal lung fibroblast HLF-1 were selected for the study.According to the different transfection substances,cells were divided into the control group(without any treatment),NC group(transfected with random sequence RNA oligo)and miR-708-5p group(transfected with mir-708-5p mimics).Cell proliferation was detected by MTT assay.Cell cycle was detected by flow cytometry.Cell invasion was detected by trans-well chamber test.Cell migration was detected by scratch test.The expression level of URGCP was detected by western blot.mRNA expression levels of miR-708-5p and URGCP were detected by real-time fluorescent quantitative PCR.Luciferase assay was conducted to verify the targeting relationship between miR-708-5p and URGCP.Results Compared with HLF-1,the expression level of miR-708-5p in A549 cells was decreased,while the expression levels of URGCP protein and mRNA were increased(P<0.05).At 72 h,the proliferation,migration and invasion abilities of the miR-708-5p group were significantly lower than those of the control and NC groups(P<0.05).Compared with the control and NC group,the expression of URGCP protein and the mRNA of URGCP gene in the miR-708-5p group were significantly decreased(all P<0.01).Compared with the WT-URGCP group,the luciferase activity of the miR-708-5p+WT-URGCP group was significantly decreased(P<0.01).Conclusion The expression level of miR-708-5p in A549 cells is decreased.Overexpression of miR-708-5p can inhibit the proliferation,migration and invasion of A549 cells,which may be related to the negative regulation of URGCP.