TGFβ signaling is a known pathway to be involved in colorectal cancer (CRC) progression and miRNAs play crucial roles by regulating different components of this pathway. Hence, finding the link between miRNAs and the pathway could be beneficial for CRC therapy. Array data indicated that miR-186-5p is a differentially expressed miRNA in colorectal Tumor/Normal tissues and bioinformatics tools predicted SMAD6/7 (inhibitory SMADs) as bona fide targets of this miRNA. Here, we intended to investigate the regulatory effect of the miR-186-5p expression on TGFβ signaling in CRC. Firstly, the miR-186-5p overexpression in HCT116 cells resulted in a significant reduction of SMAD6/7 expression, measured through RT-qPCR. Then, the direct interactions of miR-186-5p with SMAD6/7 3′UTRs were supported through dual luciferase assay. Furthermore, miR-186-5p overexpression suppressed proliferation, cell viability, and migration while, it increased apoptosis in CRC cells, assessed by cell cycle, MTT, scratch and Annexin V/PI apoptosis assays. Consistently, miR-186-5p overexpression resulted in reduced CyclinD1 protein using western blot, and also resulted in increased P21 and decreased c-Myc expression. Overall, these results introduced miR-186-5p as a cell cycle suppressor through downregulation of SMAD6/7 expression. Thus, miR-186-5p might be served as a novel tumor suppressive biomarker and therapeutic target in CRC treatment.

中文翻译：

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Hsa-miR-186-5p通过抑制结直肠癌中SMAD6和SMAD7基因的表达来调节TGFβ信号通路


TGFβ 信号传导是参与结直肠癌 (CRC) 进展的已知途径，miRNA 通过调节该途径的不同成分发挥着至关重要的作用。因此，找到 miRNA 和通路之间的联系可能有利于 CRC 的治疗。阵列数据表明，miR-186-5p 是结直肠肿瘤/正常组织中差异表达的 miRNA，生物信息学工具预测 SMAD6/7（抑制性 SMAD）是该 miRNA 的真正靶标。在这里，我们打算研究 CRC 中 miR-186-5p 表达对 TGFβ 信号传导的调节作用。首先，通过 RT-qPCR 测量，HCT116 细胞中 miR-186-5p 过表达导致 SMAD6/7 表达显着降低。然后，通过双荧光素酶测定支持 miR-186-5p 与 SMAD6/7 3'UTR 的直接相互作用。此外，通过细胞周期、MTT、划痕和膜联蛋白 V/PI 凋亡测定评估，miR-186-5p 过表达抑制增殖、细胞活力和迁移，同时增加 CRC 细胞的凋亡。一致地，使用蛋白质印迹法，miR-186-5p 过表达导致 CyclinD1 蛋白减少，并且还导致 P21 表达增加和 c-Myc 表达减少。总体而言，这些结果通过下调 SMAD6/7 表达引入 miR-186-5p 作为细胞周期抑制剂。因此，miR-186-5p可能作为结直肠癌治疗中的新型肿瘤抑制生物标志物和治疗靶点。