HAUSP-NANOG Network Involved In Gliomas

Objective To detect the expression and significance of HAUSP in differentpathological glioma tissues in vivo and glioma cell line U87in vitro, the subcellularlocation of HAUSP and NANOG in glioma cells and the expression in giloma tissues,explore their correlation in the pathogenesis of glioma and the differentiated state ingliomas of different pathological grades.To study the role of the HAUSP-NANOGregulatory network involved in gliomas biology, which lay a foundation to the further exploreits role in biological behaviour of gliomas by HAUSP-NANOG regulatory network.Methods (1)The expression of HAUSP protein was detected byimmunohistochemistry/Western blot analysis and HAUSP mRNA was examined byReal time-PCR in the pathological specimens from70cases of glioma tissues and10cases of normal brain tissues respectively.(2) The expression of HAUSP and CD133were detected by immunohistochemistry in the specimens of72cases of glioma tissues.BTSCs were isolated from glioma cell line U87and cultured in simplified serum-freeneural stem cell medium by nanosphere suspension culture method spheres, andpurified continuously through the monoclonal formation experiment. ATRA was used toinduce the differentiation of BTSCs. Double immunofluorescence staining was used todetect the co-expressions of HAUSP and CDl33.(3) The expression of HAUSP andNANOG was detected by immunohistochemical staining SP assay in72cases of humanglioma; the subcellular location in human glioma cell U87was detected by doubleimmunofluorescence staining.Results (1) The relative protein level of HAUSP was significantly different indifferent pathological grades of the human gliomas(F=15.926, P=0.000), There wassignificant difference of the relative mRNA level of HAUSP in different pathological grades of the human gliomas(F=57.892,P=0.000). The relative level of HAUSPexpression is obviously correlated with the pathological grade. Nearly negativeexpression of HAUSP was observed in normal brain tissues.(2) BTSCs were isolated,cultured and purified successfully from glioma cell line U87. They had ability ofself-renewal and proliferation, and expressed CD133, a cell surface marker for putativeNSCs. Addition of10%FBS for10days, the cells from BTS showed some degree ofdifferentiation, clearly heterogeneous cell morphology, different sizes of nuclei andwere stained positive for GFAP and NSE. The percentages of HAUSP+, CDl33+indifferent pathological levels of gliomas were significantly different (P＜0.05), and theexpressions of HAUSP+, CDl33+cells in gliomas of grade III and IV were significantlyhigher than those in glioma of gradeⅡ(P<0.05). The HAUSP+expression and CD133+expression were positively correlated (r=0.915，P=0.000). HAUSP+and CD133+expressions were reduced followed as the differentiation of BTSCs.(3) The expressionsof HAUSP and NANOG had significantly positive correlation with the grade of gliomamalignancy (P<0.05) and there was a significant correlation between the rate of HAUSPpositive cells and that of NANOG in experimental group; they were all expression inU87, and has co-location in the nucleus.Conclusion (1) HAUSP is overexpressed in gliomas. The relative level of HAUSPexpression is correlated with the pathological grade in vivo.(2) BTSCs exist in gliomacell line U87. HAUSP and stem cell marker CDl33has a significant correlation, andthey enjoy significant co-expressions in cellular level. HAUSP may play a crucial rolein the maintenance of glioma stem cells undifferentiated or poorly differentiated state.(3) HAUSP and NANOG play an important role in the pathogenesis of human gliomaand related to the malignancy; NANOG as a marker of glioma stem cells, and plays avital role in maintaining the cancer stem cells under the undifferentiated state, maybethrough the deubiquitinylation of HAUSP, which provide a basis for investigation of the role of HAUSP-NANOG regulation network and the mechanism of NANOGPosttranslational modification in gliomas in vitro and vivo.