miRNA-145通过调控IRS-1影响葡萄膜黑色素瘤细胞增殖

背景：微小RNA（miRNA）在肿瘤发生过程中可以作为癌基因或者抑癌基因发挥作用。本文中我们研究了miR-145在葡萄膜黑色素瘤发生过程中所起的作用。 方法：采用安捷伦miRNA芯片在葡萄膜黑色素瘤中检测miRNA的表达谱，并用实时定量聚合酶链式反应（RT-PCR）验证miR-145在正常葡萄膜组织，葡萄膜黑色素瘤组织和葡萄膜黑色素瘤细胞系中的表达水平。使用慢病毒表达系统构建了稳定过表达miR-145的葡萄膜黑色素瘤细胞系MUM-2B和OCM-1。使用MTT方法检测细胞增殖。使用流式细胞术分析细胞周期和细胞凋亡。通过生物信息学预测miR-145的靶基因，并通过双荧光素酶报告基因检测来验证。使用Western blot检测潜在的miR-145靶蛋白IGF-1R和IRS-1的表达。在OCM-1细胞系中敲除IRS-1基因通过TUNEL, BrdU以及流式细胞术验证其在细胞中的功能。 结果：与正常葡萄膜组织样本比较，葡萄膜黑色素瘤组织中发现了47种miRNA上调，61种miRNA下调。其中miR-145在葡萄膜黑色素瘤组织和细胞系中表达比正常葡萄膜组织显著下调。在葡萄膜黑色素瘤细胞系中过表达miR-145可以抑制细胞增殖，并将阻断细胞从G1期进入S期，同时还可以促进肿瘤细胞的凋亡。鉴定了IRS-1可能为miR-145在葡萄膜黑色素瘤中的靶基因。敲除IRS-1基因与过表达miR-145具有类似的效果。 结论：我们的数据首次表明了miR-145可能通过下调IRS-1而抑制葡萄膜黑色素瘤细胞增殖。

MicroRNA 145 may play an important role in uveal melanoma cell growth by potentially targeting IRS-1

Background. MicroRNAs (miRNAs) contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. In this study, we investigated the role of miR-145 in the pathogenesis of uveal melanoma. Methods. Expression profiles of miRNAs in uveal melanoma were performed using Agilent miRNA array. Quantitative real-time polymerase chain reaction was used to screen the expression levels of miR-145 in normal uveal tissue, uveal melanoma tissue and uveal melanoma cell lines. Lenti-virus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Cell proliferation, cell cycle and cell apoptosis of these miR-145 overexpression cell lines were examined by MTT assay and flow cytometry, rescpectively. The target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase reporter assay. The expression of insulin-like growth factor-1 receptor (IGF-1R), insulin receptor substrate-1 (IRS-1) proteins was determined by Western blot analysis. IRS-1 was knocked down in OCM-1 cells. TUNEL, BrdU and flow cytometry assay were performed in IRS-1 knocked down OCM-1 cell lines to analyze its function. Results. Fourty seven miRNAs were up-regulated in uveal melanoma and 61 were down-regulated. miR-145 expression was significantly lower in uveal melanoma sample and cell lines compared with normal uveal sample. Overexpression of miR-145 suppressed cell proliferation by blocked the G1 phase entering S phase in uveal melanoma cells, and promoted uveal melanoma cell apoptosis. IRS-1 was identified as a potential target of miR-145 by dual luciferase reporter assay. Knocking down of IRS-1 had similar effect as overexpression of miR-145. Conclusions. For the first time, we showed that miR-145 might act as a tumor suppressor in uveal melanoma, and down-regulation of the target IRS-1 might be a potential mechanism.