A Mechanistic Study Of Nucleostemin-Promoted Nucleolar Polyubiquitylation Of P27^kip1 In HCC Development

ObjectiveThe study was designed to investigate the role and mechanism of NS-promoted nucleolar polyubiquitylation of p27 during hepatocarcinogenesis,and to analyze the expression and clinical significance of NS and p27 in patients with hepatocellular carcinoma(HCC).MethodsTo screen the molecules interacting with p27 in HCC tissues.To verify the interaction between NS and p27,and clear the binding domains of the two proteins.To investigate the mechanism of NS-p27 interaction on the functions of p27 and CDK2.To study the effects of NS-p27 interaction on the proliferation of HCC cells and the growth of HCC in vivo and in vitro.To detect the expression of NS and p27in 8 fresh HCC tissues and paracancerous tissues and 118 HCC paraffin chips,and to analyze the associations of NS and p27 expression with clinicopathologic parameters and prognostic of HCC patients.Results1.IP-MS analysis indicated NS was a novel binding protein of p27 in HCC tissues.IP,GST pull-down and IF were performed to determine the direct association between NS and p27,and they were co-localizated in the nucleolus of HCC cells.2.In vitro ubiquitylation and subcellular fractionation assay suggested that NS facilitated the ubiquitylation of p27 in the nucleoli of HCC cells.IP and WB indicated NS-mediated p27 ubiquitylation could disturb the interaction between p27 and CDK2,and cause the upregulation of CDK2 and cyclin E in HCC cells.3.EdU incorporation,CCK-8 and flow cytometric analysis suggested that depletion of NS obviously resulted in proliferative impairment in HCC cells.Nude mouse tumorigenicity assay indicated knockdown of NS significantly suppressed HCC growth in vivo.4.WB and IHC suggested NS expression was elevated in HCC tissues relative to the adjacent normal ones,which was inversely correlated with that of p27(r=-0.494,p<0.01).χ~2 test indicated that NS expression was significantly associated with tumor differentiation(p=0.001),tumor metastasis(p=0.039)and AFP(p=0.020).p27 expression was associated with tumor differentiation(p=0.021)and AFP(p=0.037)in HCC patients.Finally,Kaplane-Meier analysis revealed that patients with high NS or low p27 expression was associated with worsened survival(p<0.001).Conclusions1.In hepatoma cells,NS collects p27 from the cytoplasm to the nucleolus,promotes ubiquitination of p27 and dissociates p27-CDK2 interaction in the cytoplasm,subsequently resulting in CDK2 activation and hepatoma cell proliferation.2.High NS or low p27 expression can indicate HCC diagonosis,and may represent poor prognosis for HCC patients.3.NS/p27 pathway may serve as molecular regulatory sites for HCC development,and may become a new therapeutic target of HCC.