ABSTRACT
Differential microRNA (miRNA) expression can portend clear cell renal cell carcinoma (ccRCC) progression. In a previous study, we identified a subset of dysregulated miRNA in small renal masses, pT1 ccRCC (≤5 cm) that are associated with an aggressive phenotype. The present study investigated miRNA expression in clinical stage I (cT1) tumors (≤5 cm), comparing pathologic stage I (pT1) tumors to those upstaged to pathologic stage 3 (pT3) after surgery following identification of renal vein invasion or invasion into adjacent fat tissue within Gerota's fascia. Twenty cT1 tumors were examined in an miRNA screening, 10 pT1 and 10 pT3 tumors. The ccRCC cell lines 786-O and Caki-1 were used to assess the impact of let-7c-5p and its protein target insulin-like growth factor 1 receptor (IGF1R). Cells were transfected with pre-let-7c-5p and assessed through cell proliferation, migration, and invasion assays. IGF1R expression was evaluated through Simple Western, and interaction between let-7c-5p and IGF1R was confirmed via luciferase reporter assay. Screening identified 20 miRNA, including let-7c-5p, that were dysregulated between pT1 and pT3 upstaged tumors. This miRNA was also downregulated in our previous study of pT1 tumors that progressed to metastatic disease. Transfection of ccRCC cells with pre-let-7c-5p significantly inhibited proliferation, migration, invasion, and IGF1R expression. These findings suggest that miRNA dysregulation is involved in ccRCC progression, specifically through invasion, and that let-7c-5p downregulation contributes to the aggressiveness of small ccRCC tumors, in part, through its regulation of IGF1R.
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.
ABSTRACT
BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
