MicroRNAs (miRNAs), a group of non-coding post-transcriptional regulators of gene expression, are dysregulated in clear cell renal cell carcinoma (ccRCC) and play an important role in carcinogenesis. Our prior work identified a subset of miRNAs in pT1 ccRCC tumors associated with progression to metastatic disease.
To investigate the impact of two of these dysregulated miRNA, miR-15a-5p and -26a-5p, in an effort to elucidate the mechanisms underpinning aggressive forms of stage I ccRCC.
The ccRCC cell line 786-O was transfected with pre-miRs-15a-5p and -26a-5p to rescue expression. Cell proliferation was measured via MT Cell Viability Assay. O-GlcNAc-transferase (OGT), a known protein in ccRCC proliferation, was identified by bioinformatics analysis as a target of both miRNA and validated via luciferase reporter assay to confirm binding of each miR to the 3' untranslated region (UTR). OGT protein expression was evaluated via western blotting.
Luciferase assay confirmed specificity of miR-15a-5p and -26a-5p for the OGT UTR. Western blot analysis for OGT showed reduced expression following co-transfection of both miRNAs compared to negative control or individual transfection. Co-transfection of these miRNAs greatly reduced proliferation when compared to negative control or the individual transfections.
Our results indicate that the dysregulation of miR-15a-5p and -26a-5p contribute cooperatively to the proliferation of ccRCC through their regulation of OGT. These results give insight into the pathogenesis of aggressive early stage ccRCC and suggest potential therapeutic targets for future research.
Cancer biomarkers : section A of Disease markers. 2020 Dec 03 [Epub ahead of print]
Thomas J Kalantzakos, Travis B Sullivan, Luke E Sebel, David Canes, Eric J Burks, Alireza Moinzadeh, Kimberly M Rieger-Christ
Department of Translational Research, Lahey Hospital and Medical Center, Burlington, MA, USA., Department of Urology, Lahey Hospital and Medical Center, Burlington, MA, USA., Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA, USA.