Transforming growth factor β (TGFβ) signaling pathway which is regulated by factors such as microRNAs (miRNAs) has pivotal roles in various cellular processes. Here, we intended to verify bioinformatics predicted regulatory effect of hsa-miR-5582-3P against TGFβ/SMAD signaling pathway components. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis indicated a negative correlation of expression between hsa-miR-5582-3P against TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4 putative target genes in all of tested cell lines. Also, hsa-miR-5582-3P was significantly downregulated in glioma, breast, and ovarian tumor tissues compared with their normal pairs, detected by RT-qPCR. Then dual luciferase assay supported direct interaction between this miRNA and TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4, 3' untranslated region sequences. Western blot analysis confirmed negative effect of hsa-miR-5582-3P overexpression on at least TGFβ-R1 expression. Consistently, hsa-miR-5582-3P overexpression brought about downregulation of TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4 expression in HCT-116 cell line, followed by cell cycle arrest in sub-G1 phase, detected by flow cytometry. Altogether, our data suggest that hsa-miR-5582-3P reduces the TGFβ/SMAD signaling pathway through downregulation of TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4 transcripts. These data introduce hsa-miR-5582-3P as a potential tumor suppressors-miR and a therapy candidate to be tested in cancers in which TGFβ/SMAD is deregulated.
Keywords: SMAD; dual luciferase assay; hsa-miR-5582-3P; transforming growth factor β signaling.
© 2018 Wiley Periodicals, Inc.