miRNA-4686通过靶向PDIA4影响食管癌细胞增殖和迁移

miRNA-4686 effects the proliferation and migration of esophageal cancer cells via targeting PDIA4

目的探讨miRNA-4686(miR-4686)和蛋白质二硫键异构酶A4(PDIA4)在食管癌组织和细胞中的表达,以及miR-4686体外对食管癌细胞增殖和迁移能力的影响及可能机制。方法采用miRWalk version 3在线网站预测到miR-4686与PDIA4 mRNA互补结合。利用从加州大学圣克鲁兹分校基因组(UCSC)数据库获得的miR-4686和PDIA4 mRNA的表达谱,分析食管癌组织和癌旁组织中miR-4686和PDIA4 mRNA相对表达量。从UCSC数据库获得miR-4686在人体细胞中的定位。采用实时荧光定量聚合酶链反应(qRT-PCR)检测食管癌细胞Eca109、TE-13、EC9706、KYSE-510与正常食管上皮细胞HET-1A中miR-4686和PDIA4 mRNA的相对表达量。选择miR-4686相对表达量最低的Eca109细胞进行后续研究。将Eca109细胞分为miR-4686组(转染miR-4686模拟物)和miR-NC组(转染miR-4686阴性对照序列模拟物)。通过克隆形成实验检测两组Eca109细胞增殖能力,划痕愈合实验检测Eca109细胞迁移能力,双萤光素酶报告基因实验验证miR-4686与PDIA4 mRNA的靶向关系;蛋白质印迹法检测PDIA4蛋白和PI3K-AKT-mTOR信号通路相关蛋白的表达。结果与癌旁组织比较,食管癌组织中miR-4686呈低表达、PDIA4 mRNA呈高表达(均P<0.01)。与正常食管上皮细胞HET-1A(0.98±0.15)比较,食管癌细胞Eca109(0.11±0.04)、TE-13(0.58±0.10)、EC9706(0.34±0.05)、KYSE-510(0.69±0.06)中miR-4686相对表达量均降低(均P<0.05)。与miR-NC组比较,miR-4686组Eca109细胞miR-4686相对表达量升高(9.4±2.1比1.0±0.4,t=3.88,P=0.008),克隆形成数减少[(38±9)个比(114±18)个,t=3.78,P=0.009],划痕愈合率降低[(27.13±0.91)%比(45.05±3.89)%,t=4.48,P=0.004],PDIA4 mRNA相对表达量降低[1.0±0.5比6.3±0.9,t=5.04,P=0.002]。与野生型(WT)-PDIA4+miR-NC组比较,WT-PDIA4+miR-4686组Eca109细胞相对荧光素酶活性下降(0.31±0.08比0.99±0.08,t=5.96,P<0.001)。与miR-NC组比较,miR-4686组Eca109细胞中PDIA4、p-PI3K、p-AKT、p-m-TOR蛋白表达均降低。结论食管癌组织中miR-4686呈低表达,PDIA4 mRNA呈高表达。miR-4686可能通过靶向调控PDIA4表达而抑制食管癌Eca109细胞增殖和迁移。

Objective To investigate the expressions of miRNA-4686(miR-4686)and protein disulfide isomerase A4(PDIA4)in esophageal cancer tissues,and the effect of miR-4686 on the proliferation and migration of esophageal cancer cells in vitro and possible mechanisms.Methods The complementary integration of miR-4686 and PDIA4 mRNA were predicted using the miRWalk version 3 online website.The expression profiles of miR-4686 and PDIA4 mRNA were obtained from the University of California,Santa Cruz Genome(UCSC)database,and the relative expression of miR-4686 and PDIA4 mRNA in esophageal cancer tissues and paracancerous tissues were analyzed.The UCSC database was used to obtain the localization of miR-4686 in human cells.The relative expressions of miR-4686 and PDIA4 mRNA in esophageal cancer Eca109,TE-13,EC9706 and KYSE-510 cells and normal esophageal epithelial HET-1A cells were detected by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).Eca109 cells with the lowest relative expression of miR-4686 were selected for subsequent research,and the Eca109 cells were divided into miR-4686 group(transfected with miR-4686 mimic)and miR-NC group(transfected with miR-4686 negative control sequence mimic).The proliferation ability of Eca109 cells in the two groups was detected by colony formation assay,the migration ability of Eca109 cells was detected by scratch healing assay,and the targeting relationship between miR-4686 and PDIA4 mRNA was verified by dual luciferase reporter gene assay;the expressions of PDIA4 protein and PI3K-AKT-mTOR signaling pathway-related proteins were detected by Western blotting.Results Compared with the paracancerous tissues,miR-4686 expression was low and PDIA4 mRNA expression was high in esophageal cancer tissues(both P<0.01).Compared with normal esophageal epithelial HET-1A cells(0.98±0.15),the relative expression of miR-4686 in esophageal cancer Eca109(0.11±0.04),TE-13(0.58±0.10),EC9706(0.34±0.05)and KYSE-510 cells(0.69±0.06)were all decreased(all P<0.05).Compared with the miR-NC group,the relative expression of miR-4686 in Eca109 cells of the miR-4686 group increased(9.4±2.1 vs.1.0±0.4,t=3.88,P=0.008),the number of colony formation decreased(38±9 vs.114±18,t=3.78,P=0.009),the scratch healing rate decreased[(27.13±0.91)%vs.(45.05±3.89)%,t=4.48,P=0.004],and the relative expression of PDIA4 mRNA decreased[1.0±0.5 vs.6.3±0.9,t=5.04,P=0.002].Compared with wild type(WT)-PDIA4+miR-NC group,the relative luciferase activity of Eca109 cells in WT-PDIA4+miR-4686 group decreased(0.31±0.08 vs.0.99±0.08,t=5.96,P<0.001).Compared with miR-NC group,the expressions of PDIA4,p-PI3K,p-AKT and p-m-TOR proteins in Eca109 cells of the miR-4686 group were all decreased.Conclusions In esophageal cancer tissues,miR-4686 is lowly expressed and PDIA4 mRNA is highly expressed.miR-4686 may inhibit the proliferation and migration of esophageal cancer Eca109 cells by targeting regulation of PDIA4 expression.