Expression And Significance Of NANOG In Gliomas

Objective To detect the expression and significance of NANOG in different pathological glioma tissues in vivo and glioma cell line U87 in vitro. To construct a lentiviral vector of RNAi of NANOG, which lay a foundation to the further explore its role in biological behaviour of gliomas by lentivirus-mediated silencing NANOG.Methods (1)The expression of NANOG protein was detected by immunohistochemistry/Western blot analysis and NANOG mRNA was examined by RT-PCR in the pathological specimens from 50 cases of glioma tissues and 7 cases of normal brain tissues respectively.(2) BTSCs were isolated from glioma cell line U87 and cultured in simplified serum-free neural stem cell medium by nanosphere suspension culture method spheres, and purified continuously through the monoclonal formation experiment. The immunofluorescence staining of cells was employed to identify the BTSCs and differentiated cells. The NANOG protein was detected by immunocytochemistry/Western blot analysis and expression of NANOG mRNA was examined by RT-PCR in U87 parental attached cells and BTSCs respectively. (3) The effective sequence of shRNA targeting NANOG gene was designed according to the E1bashir's criteria and the RNAi vector rule. The complementary DNA containing both sense and antisense oligo DNA of targeting sequence of NANOG was synthesized and cloned into pLKO.1-puro vector, to construct the lentiviral vector which expressed shRNA, and it was confirmed by PCR identification and DNA sequencing. The pLKO.1-NANOG-shRNA, pCMV-dR 8.2 dvpr and pCMV-VSV-G were cotransfected into 293T incasing cells. Harvesting recombinant lentivirus and detecting viral titer that was able to express shRNA targeting NANOG gene.Results (1) The relative protein level of NANOG was significantly different in different pathological grades of the human gliomas(F=65.901, P=0.000), There was significant difference of the relative mRNA level of NANOG in different pathological grades of the human gliomas(F=14.918, P=0.000). The relative level of NANOG expression is obviously correlated with the pathological grade. The NANOG protein and mRNA were not detected in normal brain tissues. (2) BTSCs were isolated, cultured and purified successfully from glioma cell line U87. They had ability of self-renewal and proliferation, and expressed CD133, a cell surface marker for putative NSCs. Addition of 10% FBS for 10 days, the cells from BTS showed some degree of differentiation, clearly heterogeneous cell morphology, different sizes of nuclei and were stained positive for GFAP and NSE. Both NANOG protein and mRNA were expressed in U87 parental attached cells and BTSCs. The relative protein level of NANOG was significantly different in U87 parental attached cells and BTSCs(t=5.719,P=0.000). There was significant difference of the relative mRNA level of NANOG in U87 parental attached cells and BTSCs(t=4.770,P=0.000). The relative level of NANOG protein and mRNA expression was higher in BTSCs than U87 parental attached cells. (3) The recombinant lentiviral vector expressing shRNA targeting NANOG gene was successfully constructed and identified by PCR and DNA sequencing. The recombinant lentivirus were harvested from supernatant of 293T cells with a viral titer of 10~6TU/ml.Conclusion (1) NANOG is overexpressed in gliomas. The relative level of NANOG expression is correlated with the pathological grade in vivo. (2) BTSCs exist in glioma cell line U87. The relative level of NANOG expression in BTSCs were higher than that in U87 parental attached cells. (3) The lentivirus RNAi vector of NANOG was constructed successfully, which provide a basis for investigation of the role of NANOG and the mechanism of NANOG transcriptional regulation in gliomas in vitro and vivo.