miRNA-744-5p靶向TGFβ-1抑制胃癌侵袭迁移的机制研究

摘要

目的 检测胃癌组织中和人胃癌细胞株中miRNA-744-5p的表达情况,并通过改变胃癌细胞株中miRNA-744-5p的表达,观察人胃癌细胞株侵袭、迁移能力的变化并探讨生物学机制.方法 通过荧光定量PCR(qRT-PCR)检测37例胃癌患者的胃癌组织及其配对的癌旁组织中miRNA-744-5p的表达水平,并对胃癌细胞株和正常胃黏膜上皮细胞株中的miRNA-744-5p表达水平进行检测.在胃癌细胞中分别转染miRNA-744-5p模拟物(mimic)、抑制物(inhibitor)以上调、下调miRNA-744-5p的表达,通过Transwell试验检测其对胃癌细胞侵袭、迁移能力的影响.将通过生物信息学软件筛选的miRNA-744-5p潜在的靶基因进行双荧光素酶试验及Western blot试验验证,并探讨其促转移的分子机制.结果 胃癌组织、胃癌细胞株的miRNA-744-5p表达水平较正常胃黏膜组织、正常胃黏膜上皮细胞株均显著降低(P<0.05).分别上调、下调胃癌细胞中miRNA-744-5p的表达水平,能够抑制、促进胃癌细胞的侵袭、迁移能力.miRNA-744-5p与TGFβ-1间存在反向调控的关系,TGFβ-1是miRNA-744-5p的靶基因.进一步实验证明,下调miRNA-744-5p的表达时,TGFβ-1表达上调,而当上调miRNA-744-5p的表达时,TGFβ-1表达下调.结论 在胃癌组织中miRNA-744-5p的表达水平降低,导致TGFβ-1在胃癌组织中的高表达以促进胃癌细胞的侵袭、迁移.%Objective To detect the miR-744-5p expression level in gastric cancer(GC) tissue and cells,and to observe the effects of invasion and migration on the gastric cancer cell due to the changes of miRNA expression. To explore the biological function of miR-744-5p in GC. Methods The miR-744-5p expression level was detected in 37 paired GC tissues and normal gastric mucosal tissues by real-time PCR. The expression levels of miR-744-5p in five GC cell lines and the normal gastric mucosal cell(GES-1) were detected similarly. SGC-7901 which with the highest expression level were transected with miR-744-5p inhibitor,and AGS which with the lowest expression level were transected with miR-744-5p mimics. Then they were selected to use for the invasion and migration tests. TGFβ-1 was predicted as a target gene of miR-744-5p by screening of the miRBase Targets,TargetScan Release 5.0 and PicTar databases and identified by the luciferase reporter assay and Western Blot. Results The expression levels of miR-744-5p were lower in GC tissues and cells than in their pair-matched adjacent normal tissues and normal gastric mucosal cell. Up-regulation and down-regulation the expression of miR-744-5p levels could restrain and promote GC cells invasion and migration ability. TGFβ-1 expression was inversely correlated with miR-186-3p expression in GC. TGFβ-1 is one of target gene of miR-744-5p. Conclusions MiR-744-5p is down-regulated in GC tissues and cell lines and associates with the metastasis of GC cell by targeting TGFβ-1.