环状RNA0026344靶向miR-21对肝癌细胞增殖、迁移、侵袭的影响及机制

Effect and mechanism of circular RNA0026344 targeting miR-21 on proliferation,migration,and invasion of liver cancer cells

目的探讨环状RNA0026344(circ_0026344)靶向微小RNA-21(miR-21)对肝癌细胞增殖、迁移、侵袭的影响及机制。方法收集25例肝癌患者术中切除的肝癌组织及其癌旁组织(距离肿瘤边缘>3 cm),用qRT-PCR检测组织中circ_0026344、miR-21表达。常规培养HCC9204细胞,将细胞随机分组并转染,分为pcDNA组(转染pcDNA)、pcDNA-circ_0026344组(转染pcDNA-circ_0026344)、anti-miR-NC组(转染anti-miR-NC)、anti-miR-21组(转染anti-miR-21)、pcDNA-circ_0026344+miR-NC组(转染pcDNA-circ_0026344+miR-NC)、pcDNA-circ_0026344+miR-21组(转染pcDNA-circ_0026344+miR-21 mimics)。转染48 h后用RT-qPCR检测各组circ_0026344、miR-21表达,确认转染效率。用MTT法检测细胞增殖能力,用平板克隆实验检测细胞克隆形成数,用Transwell实验检测细胞迁移和侵袭能力,双荧光素酶报告实验验证circ_0026344与miR-21的靶向关系,Western blotting法检测细胞内上皮间充质转化(EMT)相关蛋白(E-cadherin、N-cadherin)表达。结果与癌旁组织比较,肝癌组织中circ_0026344相对表达量低,而miR-21相对表达量高(P均<0.05)。与pcDNA组比较,pcDNA-circ_0026344组HCC9204细胞增殖能力、克隆形成数、迁移及侵袭细胞数、N-cadherin蛋白相对表达量低(P均<0.05),E-cadherin蛋白相对表达量高(P<0.05)。Circular RNA Interactome数据库在线分析显示,miR-21与circ_0026344存在特异性结合位点。与miR-NC+wt-circ_0026344共转染比较,miR-21 mimics+wt-circ_0026344共转染后细胞相对荧光素酶活性低(P<0.05);而与miR-NC+mut-circ_0026344共转染比较,miR-21 mimics+mut-circ_0026344共转染后细胞相对荧光素酶活性差异无统计学意义(P>0.05)。与anti-miR-NC组比较,anti-miR-21组HCC9204增殖能力、克隆形成数、迁移和侵袭细胞数、N-cadherin蛋白相对表达量低(P均<0.05),E-cadherin蛋白相对表达量高(P<0.05)。与pcDNA-circ_0026344+miR-NC组比较,pcDNA-circ_0026344+miR-21组增殖能力、克隆形成数、迁移和侵袭细胞数、N-cadherin蛋白相对表达量高(P均<0.05),E-cadherin蛋白相对表达量低(P<0.05)。结论circ_0026344可靶向miR-21抑制肝癌细胞增殖、迁移、侵袭,其作用机制可能与调控EMT有关。

Objective To explore the effect and mechanism of circular RNA0026344(circ_0026344)targeting microRNA-21(miR-21)on the proliferation,migration and invasion of liver cancer cells.Methods The exression of circ_0026344 and miR-21 was detected by qRT-PCR in the liver cancer tissues and adjacent tissues(3 cm from the tumor edge)resected from 25 patients with hepatocellular carcinoma.HCC9204 cells were routinely cultured and randomly divided into the pcDNA group(transfected with pcDNA),pcDNA-circ_0026344 group(transfected with pcDNA-circ_0026344),anti-miR-NC group(transfected with anti-miR-NC),anti-miR-21 group(transfected with anti-miR-21),pcDNA-circ_0026344+miR-NC group(transfected with pcDNA-circ_0026344+miR-NC),and pcDNA-circ_0026344+miR-21 group(transfected with pcDNA-circ_0026344+miR-21 mimics).The circ_0026344 and miR-21 expression levels were detected by qRT-PCR at 48 h after transfection.Cell proliferation ability was detected by MTT assay,cell clone formation number was detected by plate cloning assay,cell migration and invasion abilities were detected by Transwell assay,and the targeting relationship between circ_0026344 and miR-21 was verified by dual luciferase report assay.The expression of EMT-related proteins(E-cadherin and N-cadherin)was detected by Western blotting.Results Compared with the adjacent tissues,the relative expression level of circ_0026344 in liver cancer tissues was lower,while the relative expression level of miR-21 was higher(both P<0.05).Compared with the pcDNA-circ_0026344 group,the proliferation ability,clone formation number,migration and invasion number of HCC9204 cells and the relative expression of N-cadherin protein in the pcDNA-circ_0026344 group were lower(all P<0.05),and the relative expression of E-cadherin protein was higher(P<0.05).Circular RNA Interactome database online analysis showed that there was a specific binding site between miR-21 and circ_0026344.Compared with the co-transfection of miR-NC+wt-circ_0026344,the relative luciferase activity of cells after co-transfection of miR-21 mimics+wt-circ_0026344 was lower(P<0.05);while there was no significant difference in the relative luciferase activity between cells after co-transfection with miR-21 mimics+mut-circ_0026344 and cells after co-transfection of miR-NC+mut-circ_0026344(P>0.05).Compared with the anti-miR-NC group,the proliferation ability,clone formation number,migration and invasion number of HCC9204 cells and the relative expression of N-cadherin protein in the anti-miR-21 group were lower(all P<0.05),and the relative expression of E-cadherin protein was higher(P<0.05).Compared with the pcDNA-circ_0026344+miR-NC group,the proliferation ability,clone formation number,migration and invasion number of HCC9204 cells and the relative expression of N-cadherin protein in the pcDNA-circ_0026344+miR-21 group were higher(all P<0.05),while the relative expression of E-cadherin protein was lower(P<0.05).Conclusion The circ_0026344 targeting miR-21 can inhibit the proliferation,migration and invasion of liver cancer cells by regulating EMT.