MiRNA-424-5p Suppresses Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma and Attenuates Expression of O-GlcNAc-Transferase

SIMPLE SUMMARY: The identification of biomarkers that predict the metastatic potential of tumors is a current area of interest in cancer research. A previous study from our laboratory identified numerous microRNA (miRNA) biomarkers that are differentially expressed in pathologic stage I clear cell renal cell carcinoma (ccRCC) tumors that progress to metastatic disease. This study investigated the role of aberrant expression of one of these miRNA, miR-424-5p, and one of its associated protein targets, O-GlcNAc-transferase (OGT). We examined the influence of miR-424-5p and OGT expression on the proliferation, migration, and invasion of ccRCC cells, and confirmed the direct interaction between miR-424-5p and OGT. These findings suggest that the decrease in miR-424-5p expression observed in these small renal masses leads to an increase in OGT, which facilitates metastasis. ABSTRACT: MicroRNAs (miRNAs) are non-coding post-transcriptional regulators of gene expression that are dysregulated in clear cell renal cell carcinoma (ccRCC) and play an important role in tumor progression. Our prior work identified a subset of miRNAs in pT1 ccRCC tumors, including miR-424-5p, that are associated with an aggressive phenotype. We investigate the impact of this dysregulated miRNA and its protein target O-GlcNAc-transferase (OGT) to better understand the mechanisms behind aggressive stage I ccRCC. The ccRCC cell lines 786-O and Caki-1 were used to assess the impact of miR-424-5p and OGT. Cells were transfected with pre-miR-424-5p, a lentiviral anti-OGT shRNA, or were treated with the demethylating agent 5-Aza-2′-deoxycytidine. Cell proliferation was measured via MT cell viability assay. Cell migration and invasion were analyzed using Transwell assays. The expression of miR-424-5p was determined through qRT-PCR, while OGT protein expression was evaluated through Western blotting. The interaction between miR-424-5p and OGT was confirmed via luciferase reporter assay. The transfection of ccRCC cells with pre-miR-424-5p or anti-OGT shRNA significantly inhibited cell proliferation, migration, and OGT expression, while miR-424-5p also attenuated cell invasion. Addition of the demethylating agent significantly reduced cell proliferation, migration, invasion, and OGT expression, while significantly increasing the expression of miR-424-5p. Altogether, these findings suggest that epigenetic downregulation of miR-424-5p, which in turn augments OGT expression, contributes to the creation of aggressive forms of stage I ccRCC.