INTRODUCTION & OBJECTIVES: MicroRNAs (miR) are small, noncoding RNAs that regulate gene expression and are involved in different biological processes including differentiation, proliferation and apoptosis. Mutations or altered expression of miRs might cause several diseases including cancer. Since a variable expression of miR501 not related with the patient age or sex in 63 pairs of normal and clear cell renal carcinoma (ccRCC) tissues has been found, we have analysed the possible function of this miR in ccRCC. MATERIAL & METHODS: Analysis of miR501 expression was performed by microarray and real time RT-PCR. miR501 up or downregulation was performed by cell transfection with a specific plasmid expressing miR501 sequences (PL-501) and antagomiR, respectively. Apoptosis was studied through caspase-3 activity and cell cycle analysis. Cell proliferation was evaluated by direct cell count and with the CellTiter method. Protein levels and kinase activity were calculated by using immunological techniques and cell imaging. RESULTS: Follow up analysis at least 5 years in 35 ccRCC subjects showed a good prognosis for patients with a lower expression (<1) of miR501 in ccRCC tissues compared with normal renal parenchyma. Conversely, 50% of patients with unchanged or higher levels of miR501 exhibited a poor prognosis with a 25% of deaths. In order to evaluate the role of miR501 in renal cancer, we have modified its expression transfecting kidney carcinoma cells KJ29 with a specific antagomiR and with the PL-501 plasmid. MiR501 downregulation caused a reduction of mTOR activity, the increase of G0/G1 phase of cell cycle and induced apoptosis by enhancing the activity of caspase-3. Activation of apoptosis occurred in a p53-dependent manner without affecting the expression of the mTOR-related MDM2 protein, an inhibitor of p53, which results overexpressed in metastatic kidney carcinoma. On the contrary, miR501 upregulation caused mTOR activation, increased expression of MDM2 and enhanced cell proliferation and survival. CONCLUSIONS: These findings suggest for the miR501 a role of kingmaker among apoptosis and cell survival in ccRCC patients. When this miR is downregulated it stimulates apoptosis, but if shows unchanged or higher levels compared with control it promotes the cell growth. This hypothesis is also consistent with follow up data, therefore, the expression of miR501-5p could be considered as a new biomarker for the prognosis of clear cell renal carcinoma.
MicroRNA501 may affect the aggressiveness of clear cell renal carcinoma
BONON, Anna;MANGOLINI, Alessandra;LANZA, Giovanni;RUSSO, Gian Rosario;AGUIARI, Gianluca
2014
Abstract
INTRODUCTION & OBJECTIVES: MicroRNAs (miR) are small, noncoding RNAs that regulate gene expression and are involved in different biological processes including differentiation, proliferation and apoptosis. Mutations or altered expression of miRs might cause several diseases including cancer. Since a variable expression of miR501 not related with the patient age or sex in 63 pairs of normal and clear cell renal carcinoma (ccRCC) tissues has been found, we have analysed the possible function of this miR in ccRCC. MATERIAL & METHODS: Analysis of miR501 expression was performed by microarray and real time RT-PCR. miR501 up or downregulation was performed by cell transfection with a specific plasmid expressing miR501 sequences (PL-501) and antagomiR, respectively. Apoptosis was studied through caspase-3 activity and cell cycle analysis. Cell proliferation was evaluated by direct cell count and with the CellTiter method. Protein levels and kinase activity were calculated by using immunological techniques and cell imaging. RESULTS: Follow up analysis at least 5 years in 35 ccRCC subjects showed a good prognosis for patients with a lower expression (<1) of miR501 in ccRCC tissues compared with normal renal parenchyma. Conversely, 50% of patients with unchanged or higher levels of miR501 exhibited a poor prognosis with a 25% of deaths. In order to evaluate the role of miR501 in renal cancer, we have modified its expression transfecting kidney carcinoma cells KJ29 with a specific antagomiR and with the PL-501 plasmid. MiR501 downregulation caused a reduction of mTOR activity, the increase of G0/G1 phase of cell cycle and induced apoptosis by enhancing the activity of caspase-3. Activation of apoptosis occurred in a p53-dependent manner without affecting the expression of the mTOR-related MDM2 protein, an inhibitor of p53, which results overexpressed in metastatic kidney carcinoma. On the contrary, miR501 upregulation caused mTOR activation, increased expression of MDM2 and enhanced cell proliferation and survival. CONCLUSIONS: These findings suggest for the miR501 a role of kingmaker among apoptosis and cell survival in ccRCC patients. When this miR is downregulated it stimulates apoptosis, but if shows unchanged or higher levels compared with control it promotes the cell growth. This hypothesis is also consistent with follow up data, therefore, the expression of miR501-5p could be considered as a new biomarker for the prognosis of clear cell renal carcinoma.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.