Hsa-miR-5195-3P induces downregulation of TGFβR1, TGFβR2, SMAD3 and SMAD4 supporting its tumor suppressive activity in HCT116 cells.

Abstract

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.