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, is emerging the necessity to use miRs as biological biomarkers 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 normals of the same patients. MATERIAL 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 with showed lower expression of miR-501 in cancer tissues 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 induced an increase in G0/G1 phase of cell cycle and a decrease of mTOR activity in KJ29 cells. In addition, the treatment with antagomiR caused an increase in caspase-3 activity, suggesting that this miR may regulate apoptosis. Moreover, miR-501-5p depletion enhanced the expression of p53, data also observed in kidney cancer tissues expressing lower levels of this miR than controls. The activation of p53 was also observed by its nuclear translocation in KJ29 treated with antagomiR. KJ29 cells were also transfected with a plasmid expressing miR-501-5p sequences and these cells showed an increased level of miR-501 compared with untranfected cells. CONCLUSIONS: Our findings show that miR501-5p was differentially expressed in ccRCC. High or unchanged levels of miR501-5p seem not related with grading and metastasis in ccRCC, however, when it is downregulated could promote a good prognosis. Data reported suggest an anti apoptotic role for miR501-5p, making it a likely risk factor for a poor prognosis in renal carcinoma. Therefore, the expression of miR501-5p could be considered as potential biomarker for the prognosis of clear cell kidney carcinoma. 1. Schaefer A, Stephan C, Busch J, Yousef GM, Jung K: Diagnostic, prognostic and therapeutic implications of microRNAs in urologic tumors. Nat Rev Urol;7(5):286-97, 2010. 2. Cairns P: Renal cell carcinoma. Cancer Biomark; 9(1-6):461-73, 2010. 3. Del Senno et al. Cell Biology International Reports. 1986; 10:195.