Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Int J Biochem Cell Biol. 2019 Apr;109:1-7. doi: 10.1016/j.biocel.2019.01.001. Epub 2019 Jan 16.
MicroRNAs are classified as small non-coding RNAs that regulate gene expression mainly through targeting the 3'UTR region of mRNAs. A great number of miRNAs play important role in the regulation of signaling pathways in normal and cancer cells. Here, we predicted hsa-miR-5195-3p (miR-5195-3p) as a potential regulator of TGFβ signaling and investigated its effect on TGFB-R1, TGFB-R2, SMAD2, SMAD3 and SMAD4 transcripts which are key players of TGFβ/SMAD signaling pathway. Overexpression of miR-5195 in HCT116 cells resulted in a significant reduction of TGFB-R1, SMAD2, SMAD3, and SMAD4 at the mRNA level which was confirmed using RT-qPCR. Consistently, western blot analysis confirmed that miR-5195 overexpression in HCT116 cells resulted in downregulation of TGFBR1 at the protein level. Furthermore, dual luciferase analysis verified the direct interaction of miR-5195 with TGFB-R1 and SMAD4 3'UTR sequences in SW480 cells. Additionally, flow cytometry analysis confirmed that miR-5195 overexpression significantly increased the sub-G1 and decreased the G-1 cell populations in both SW480 and HCT116 cell lines. Finally, miR-5195 overexpression significantly downregulated c-MYC and cyclin D1 but upregulated p21 genes. Overall, our results indicated that miR-5195 modulates TGFβ signaling pathway and affects the cell cycle progression through targeting TGFB-R1, TGFB-R2, SMAD2, SMAD3, SMAD4 transcripts.
微小 RNA（miRNA）被归类为小的非编码 RNA，主要通过靶向 mRNAs 的 3'UTR 区域来调节基因表达。大量的 miRNA 在正常和癌细胞中信号通路的调节中发挥重要作用。在这里，我们预测 hsa-miR-5195-3p（miR-5195-3p）作为 TGFβ 信号的潜在调节剂，并研究了其对 TGFB-R1、TGFB-R2、SMAD2、SMAD3 和 SMAD4 转录本的影响，这些转录本是 TGFβ/SMAD 信号通路的关键参与者。在 HCT116 细胞中过表达 miR-5195 导致 TGFB-R1、SMAD2、SMAD3 和 SMAD4 的 mRNA 水平显著降低，这通过 RT-qPCR 得到了证实。一致地，western blot 分析证实，在 HCT116 细胞中过表达 miR-5195 导致 TGFBR1 蛋白水平下调。此外，双荧光素酶分析验证了 miR-5195 与 TGFB-R1 和 SMAD4 3'UTR 序列在 SW480 细胞中的直接相互作用。此外，流式细胞术分析证实，在 SW480 和 HCT116 细胞系中过表达 miR-5195 显著增加了 sub-G1 期和减少了 G-1 期细胞群体。最后，miR-5195 过表达显著下调了 c-MYC 和 cyclin D1，但上调了 p21 基因。总体而言，我们的结果表明，miR-5195 通过靶向 TGFB-R1、TGFB-R2、SMAD2、SMAD3、SMAD4 转录本调节 TGFβ 信号通路并影响细胞周期进程。
