miR-4645-5p通过靶向MUC16调控食管癌细胞的恶性生物学行为

miR-4645-5p regulates malignant biological behavior of esophageal cancer cells by targeting MUC16

目的 探讨微小RNA(miR)-4645-5p靶向黏蛋白16(MUC16)对食管癌细胞增殖、侵袭、上皮间充质转化的影响及其分子机制。方法 通过TCGA数据库在线分析miR-4645-5p在食管癌组织中的表达。利用实时荧光定量聚合酶链反应(qPCR)分析食管癌细胞系中miR-4645-5p的表达水平。通过脂质体转染技术将miR-4645-5p模拟物和阴性对照模拟物转染KYSE-30细胞,作为miR-4645-5p组和对照模拟物组。分别采用CCK-8法、划痕实验、Transwell实验分析转染KYSE-30细胞的增殖能力、迁移能力和侵袭能力。通过生物信息学网站预测miR-4645-5p的靶基因,双荧光素酶报告基因实验检测miR-4645-5p对靶基因的结合。采用qPCR检测MUC16 mRNA的表达水平,Western blotting检测MUC16、转录因子-1(ZEB-1)、闭锁小带蛋白-1(ZO-1)、紧密连接蛋白-1(Claudin-1)、α-平滑肌肌动蛋白(α-SMA)蛋白的表达水平。结果 食管癌组织中miR-4645-5p表达水平低于癌旁组织(P<0.01)。与HET-1A比较,食管癌细胞系中miR-4645-5p呈低表达(P<0.05)。miR-4645-5p过表达后,KYSE-30细胞增殖能力降低(P<0.05),迁移能力降低(P<0.01),侵袭能力明显降低(P<0.01)。miR-4645-5p靶向负调控MUC16 mRNA的表达(P<0.01)。miR-4645-5p过表达后,MUC16、ZEB-1、α-SMA蛋白表达均下调,ZO-1、Claudin-1蛋白表达均上调。结论 miR-4645-5p通过靶向MUC16调控食管癌KYSE-30细胞的恶性生物学行为。

Objective To investigate the effect of microRNA(miR)-4645-5p on the proliferation,invasion and epithelial-mesenchymal transition of esophageal cancer cells by targeting mucin 16(MUC16)and its molecular mechanism.Methods The expression of miR-4645-5p in esophageal cancer tissues was analyzed online by TCGA database.The expression level of miR-4645-5p in esophageal cancer cell lines was analyzed by fluorescent real-time fluorescence quantitative polymerase chain reaction(qPCR).KYSE-30 cells were transfected with miR-4645-5p mimic and negative control mimic by lipofection technology,and were divided into miR-4645-5p group and control mimic group.The proliferation ability,migration ability and invasion ability of transfected KYSE-30 cells were analyzed by CCK-8 method,scratch test and Transwell test respectively.The target gene of miR-4645-5p was predicted by the bioinformatics website,and the binding of miR-4645-5p to the target gene was detected by the dual-luciferase reporter gene assay.The expression level of MUC16 mRNA was detected by qPCR,and the protein expression levels of MUC16,transcription factor-1(ZEB-1),zonal atresia protein(ZO-1),tight junction protein-1(Claudin-1)andα-smooth muscle actin(α-SMA)were detected by Western blotting.Results The expression level of miR-4645-5p in esophageal cancer tissues was significantly lower than that in adjacent tissues(P<0.01).Compared with HET-1A,the expression of miR-4645-5p was lower in esophageal cancer cell lines(P<0.05).After overexpression of miR-4645-5p,the proliferation ability of KYSE-30 cells was significantly reduced(P<0.05),the migration ability was significantly reduced(P<0.01)and the invasion ability was significantly reduced(P<0.01).miR-4645-5p targeted and negatively regulated the expression of MUC16 mRNA(P<0.01).After overexpression of miR-4645-5p,the protein expression levels of MUC16,ZEB-1 andα-SMA were all down-regulated,and the protein expression levels of ZO-1 and Claudin-1 were up-regulated.Conclusion miR-4645-5p regulates the malignant biological behavior of esophageal cancer KYSE-30 cells by targeting MUC16.