MiR501-5p may promote proliferation and survival in clear cell kidney carcinoma

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression and are involved in the control of several biological processes such as proliferation differentiation and apoptosis. The abnormal expression and mutation of miRs is implicated in many hereditary and neoplastic diseases including kidney carcinoma. In the last years, is emerging the necessity to use microRNA 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 that are resistant to conventional treatments. We have found that the expression of miR501-5p was higher in 35 clear cell carcinoma (ccRCC) and lower in 9 papillary carcinoma (pRCC) tissues compared with their corresponding normal kidney parenchyma. The expression levels of miR501-5p were not associated with the age of patients or tumor grading in ccRCC and, showed a variable distribution. However, patients with a low expression of miR501-5p exhibited a good prognosis compared with patient with unchanged or high levels of this miR. In order to evaluate the possible role of miR501-5p in renal cancer, we have downregulated and upregulated it by transfection of kidney carcinoma cells (KJ29) with a specific antagomiR and with a plasmid expressing miR501-5p sequences, respectively. The reduction of miR501-5p induced an increase in G0/G1 phase and a decrease of mTOR activity. In addition, the treatment with antagomiR showed an increase in both p53 expression and caspase-3 activity. Consistently, the activation of p53 was demonstrated also by its nuclear translocation. On the contrary, the overexpression of miR501-5p by transfection of KJ29 cells with the plasmid expressing this miR caused the activation of mTOR, the increased expression of MDM2 protein, an inhibitor of p53, and, the reduction of p53 expression. The inhibition of p53 occurred by its degradation through protein ubiquitination and activation of proteasoma machinery which induced a marked increase of cell survival. Taken together, these findings show that the overexpression of miR501-5p may increase cell survival, therefore an high expression of this MicroRNA could be a risk factor for a poor prognosis of renal carcinoma. Conversely, a low expression of miR501-5p seems promote a good prognosis in clear cell kidney carcinoma patients. Thus, the expression of miR501-5p could be used as new potential biomarker for the prognosis of clear cell renal carcinoma

Differential expression of microRNA501-5p affects the aggressiveness of clear cell renal carcinoma

AbstractRenal cell carcinoma is a common neoplasia of the adult kidney that accounts for about 3% of adult malignancies. Clear cell renal carcinoma is the most frequent subtype of kidney cancer and 20–40% of patients develop metastases. The absence of appropriate biomarkers complicates diagnosis and prognosis of this disease. In this regard, small noncoding RNAs (microRNAs), which are mutated in several neoplastic diseases including kidney carcinoma, may be optimal candidates as biomarkers for diagnosis and prognosis of this kind of cancer. Here we show that patients with clear cell kidney carcinoma that express low levels of miR501-5p exhibited a good prognosis compared with patients with unchanged or high levels of this microRNA. Consistently, in kidney carcinoma cells the downregulation of miR501-5p induced an increased caspase-3 activity, p53 expression as well as decreased mTOR activation, leading to stimulation of the apoptotic pathway. Conversely, miR501-5p upregulation enhanced the activity of mTOR and promoted both cell proliferation and survival. These biological processes occurred through p53 inactivation by proteasome degradation in a mechanism involving MDM2-mediated p53 ubiquitination. Our results support a role for miR501-5p in balancing apoptosis and cell survival in clear cell renal carcinoma. In particular, the downregulation of microRNA501-5p promotes a good prognosis, while its upregulation contributes to a poor prognosis, in particular, if associated with p53 and MDM2 overexpression and mTOR activation. Thus, the expression of miR501-5p is a possible biomarker for the prognosis of clear cell renal carcinoma

An ENDOR study of methyl dynamics at low temperature

We have studied the motion of the methyl group in the free radical obtained by gamma-irradiation of 4-methyl-2,6-di-t-butylphenol. The ENDOR spectra have been measured in thetemperature range 6 to 200 K. The height of hindering potential barrier has been obtained from the shift of the ENDOR lines at low temperature

EPR AND ENDOR RELAXATION STUDY OF MOLECULAR MOTIONS OF A KETONE UREA INCLUSION COMPOUND

The radical obtained by gamma irradiation of the 10-nonadecanone/urea inclusion compound has been studied by electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy. The EPR line widths have been analyzed, and the characteristics and the rates of the molecular motions of the included molecule have been obtained in the temperature range 160-290 K. Two different types of motion have been detected: the wobbling motion of the methylene groups about the carbon-carbon bond modulates the beta proton isotropic hyperfine interactions, while overall uniaxial rotation inside the host channel mainly modulates the anisotropic dipolar interaction of the alpha proton. The internal motion is faster and has higher activation energy than molecular rotation. These results have been confirmed by the analysis of the amplitudes of the ENDOR lines of the gamma and zeta protons, which shows that the internal motion rate is just in the range of the electron spin Larmor frequency, while the rotation rate is lower

AN ENDOR STUDY OF THE TEMPERATURE-DEPENDENCE OF METHYL TUNNELING

We have studied the motion of the methyl group in the free radical obtained by gamma-irradiation of 4-methyl-2,6-di-t-butylphenol (MDBP). The ENDOR spectra have been measured in the temperature range 4.5 to 200 K. The positions of the methyl proton lines are temperature dependent. Their variation is accounted for by considering the transition from the quantum regime of motion of the methyl group at low temperature to the classic regime at higher temperature. This transition is found to occur in a temperature range narrower than that anticipated by Allen's theory

ENDOR spectroscopy: a tool for the study of radical structures and motions in the solid state.

We show that the analysis of ENDOR lineshapes and enhancements can give valuable information on the dynamics of radicals in the solid state. The basic advantage of ENDOR with respect to EPR spectroscopy resides in its greater spectral resolution, which positively affects also its potentiality in studying molecular motions in solids. In fact, we show that the dynamical effects on the lineshapes are detectable by using ENDOR spectroscopy in a larger range of correlation times. The range can be still more wide if the radical dynamics gives rise to an efficient relaxation mechanism which influences the spin populations. In this case, the motions can be studied through the analysis of the ENDOR enhancements, which in some cases became a very sensitive probe

MICRORNA501 UP-REGULATION MAY INCREASE THE AGGRESSIVENESS OF CLEAR CELL RENAL CARCINOMA THROUGH MTOR ACTIVATION AND P53 DEGRADATION

Introduction: Many biological processes as gene expression, cell proliferation, differentiation and apoptosis are affected by microRNAs (miR) which are small noncoding RNAs that act at post transcriptional level. In fact, the impaired function of these short RNAs might cause several diseases including cancer (1). We have found a variable expression of miR501 in 63 pairs of normal and clear cell renal carcinoma (ccRCC) tissues, therefore, a possible function of this miR in ccRCC was investigated. Material and Methods: Analysis of miR501 expression was carried out 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 analysed through caspase-3 activity, Hoechst method and cell cycle analysis. Cell proliferation was evaluated by direct cell count and with the CellTiter method. Gene expression, protein ubiquitination and kinase activity were analysed by immunological techniques and cell imaging. Results: Follow up analysis of 35 ccRCC subjects showed a good prognosis for patients which a lower expression of miR501 in ccRCC tissues compared with normal renal parenchyma. Conversely, about the 50% of patients with unchanged or higher levels of miR501 exhibited a poor prognosis. In order to evaluate the role of miR501 in renal cancer, we have modified its expression transfecting kidney carcinoma cells KJ29 (2) 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 (3). On the other hand, miR501 upregulation caused mTOR activation that stimulated cell proliferation as well as cell survival. The latter biological processes were associated with an increased expression of MDM2 which induced p53 degradation activating the proteasome by p53 poly-ubiquitination. Discussion and conclusion: MiR501 seems to act as a molecular switch able to turn on or off mTOR signalling. In fact, the downregulation of miR501 led to sequential mTOR kinase inhibition, p53 activation and increased apoptosis. Conversely, miR501 upregulation caused mTOR activation, increased expression of MDM2 and p53 degradation, promoting cell survival, as already observed for follow up data. These findings support the role of kingmaker for the miR501 among apoptosis and cell survival in ccRCC patients, therefore miR501 expression could be used to evaluate the prognosis of patients with clear cell renal carcinoma. 1. Di Leva G, Croce CM: Roles of small RNAs in tumor formation. Trends Mol Med. 16(6):257-67, 2010. Review. 2. Barletta C et al.: Cytogenetic, molecular and phenotypic characterization of the newly established renal carcinoma cell line KJ29. Evidence of translocations for chromosomes 1 and 3. Anticancer Res. 15(5B):2129-36, 1995. 3. Noon AP et al: Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy. BJU Int. 109(8):1250-7, 2012

MiR501-5p stimulating the activity of mToR may result a risk factor for a poor prognosis of clear cell kidney carcinoma

INTRODUCTION & OBJECTIVESS: MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression and are involved in the control of several biological processes such as proliferation differentiation and apoptosis. Abnormal expression and mutation of miRs is implicated in many hereditary and neoplastic diseases including kidney carcinomas. In the last years, is emerging the necessity to use microRNA 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 that are resistant to conventional treatments. MATERIAL & 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 by a plasmid expressing miR501-5p specific sequences, respectively. Analysis of proteins was performed by immunological techniques and cell imaging. Apoptosis and cell cycle were studied by analysis of caspase-3 activity and flow cytometry after DNA staining with propidium iodide, respectively. RESULTS: Follow up data of 25 ccRCC and 5 pRCC patients showed that subjects with low expression of miR501-5p exhibited a good prognosis compared with patients with unchanged or high levels of this microRNA. 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. 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. Consistently, the upregulation of miR501-5p by transfection of KJ29 cells with a plasmid expressing miR501-5p sequences, caused an increase of mTOR activity compared with untransfected cells. The reduction of miR501-5p expression also induced an increased activity of caspase-3, likely in a p53-dependent manner. In fact, miR501-5p depletion enhanced the expression and nuclear translocation of p53 in KJ29 cells. CONCLUSIONS: Our findings suggest that miR501-5p overexpression could induce a resistance to apoptosis and therefore it could be a risk factor for a poor prognosis of renal carcinoma. In fact, lower expression of miR501-5p seems promote a good prognosis in clear cell kidney carcinoma patients. Furthermore, the expression of miR501-5p in combination with mTOR activity could be used as new potential biomarker for the prognosis of renal carcinomas

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