Downregulated miR-495-3p in colorectal cancer targets TGFβR1, TGFβR2, SMAD4 and BUB1 genes and induces cell cycle arrest

Background: Hsa-miR-495 (miR-495) has been extensively investigated in cancer initiation and progression. On the other hand, our bioinformatics analysis suggested that miR-495 exerts its effects through targeting of TGFO signaling components. Methods & Results: In order to investigate such an effect, miR-495 precursor was overexpressed in HEK293T, SW480, and HCT116 cells, which was followed by downregulation of TGFOR1, TGFOR2, SMAD4, and BUB1 putative target genes, detected by RT-qPCR. Also, luciferase assay supported the direct interaction of miR-495 with 3 ' UTR sequences of TGFOR1, TGFOR2, SMAD4, and BUB1 genes. Furthermore, a negative correlation of expression between miR-495-3p and some of these target genes was deduced in a set of colorectal and breast cancer cell lines. Then, flow cytometry analysis showed that the overexpression of miR-495 in HCT116 and HEK293T resulted in an arrest at the G1 phase. Consistently, western blotting analysis showed a significant reduction of the Cyclin D1 protein in the cells overexpressing miR-495, pointing to downregulation of the TGFO signaling pathway and cell cycle arrest. Finally, microarray data analysis showed that miR-495-3p is significantly downregulated in colorectal tumors, compared to the normal pairs. Conclusions: Overall, the results of the current study introduced miR-495-3p as a cell cycle progression suppressor, which may negatively regulate TGFOR1, TGFOR2, SMAD4, and BUB1 genes. This finding suggests miR495-3p as a tumor suppressor candidate for further evaluation.