MIR-501 DEPLETION INDUCES CELL CYCLE INHIBITION BY MTOR AND P53 MODULATION IN RENAL CARCINOMA

Introduction: MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression at post-transcriptional level. The abnormal expression and mutation of miRs has been observed in most urologic cancers including renal cancer, thus they may contribute to development and progression of kidney carcinoma. In fact, their impaired function could trigger a series of altered signalling resulting in abnormal differentiation, proliferation and apoptosis. In the last years, the necessity to use miRs as biological biomarkers is emerging in order to improve diagnosis, prognosis and therapy response in renal carcinomas. Furthermore, miRNAs might be potential targets for novel therapeutic strategies, especially in patients with tumour subtypes that do not respond to currently available therapies (1, 2). Here, we have focalized our study on the role of miR501-5p in kidney carcinomas because it has been found differently expressed in kidney cancer tissues compared with the normal kidney of the same patients. Materials and Methods: Analysis of miR501-5p expression was performed by real time RT-PCR. Depletion or enrichment of this miR was conducted by specific antagomiRs and plasmid expressing miR-501-5p specific sequences, respectively. Protein activity was analyzed by immunological and cell imaging techniques. Apoptosis was studied through caspase-3 activity and cell cycle analysis was performed by propidium iodide staining. Results: We have analyzed the expression of miR501-5p in 36 clear cell (ccRCC) and 11 papillary (pRCC) kidney carcinomas. The expression of miR501-5p was higher in ccRCC (3.72 fold) and lower (3.76 fold) in pRCC tissues compared with normal kidneys derived from the same subjects, respectively. However, the distribution of miR501-5p expression values in ccRCC was found strongly variable. Follow up data of 25 ccRCC and 5 pRCC patients suggest that subjects who showed lower expression of miR-501 in cancer tissues with respect to control (normal kidney), exhibited a good prognosis compared with patients with unchanged or high levels of this small RNA. In order to evaluate the role of miR501-5p in renal cancer, we have depleted it by a specific antagomiR in KJ29 kidney cancer cell line (3). KJ29 cells expressed higher levels of miR501-5p than normal immortalized tubular kidney cells. The transfection of KJ29 cells with antagomiR caused a 50% reduction of miR501-5p expression compared with untransfected cells. Furthermore, the reduction of miR501-5p induc