| BackgroundGlioma is a primary intracranial malignancy,which is often blurrily demarcated from surrounding tissues and is highly aggressive,resulting in recurrence,increased drug resistance and poor prognosis.The current standardized treatment protocol is surgical resection combined with postoperative radiotherapy and chemotherapy.Nevertheless,the median survival of patients with glioma is only 14.6 months.The poor therapeutic effect of glioma is mostly related to the high invasiveness.Epithelial-mesenchymal transformation(EMT)is involved in the migration and invasion process of various tumors and is closely related to the migration and invasion ability of tumors.Therefore,targeting glioma's EMT is of great significance to enhance the therapeutic effect of glioma and improve the prognosis of glioma patients.Zinc finger proteins,which can be involved in several aspects of glioma as oncogenes or antioncogenes,and can affect the prognosis of glioma patients.The role of zinc finger protein family members in the glioma EMT has been reported previously.While the effect and mechanism of zinc finger protein 263(ZNF263)on EMT in glioma has yet to be explored.ObjectiveIn this study,we aim to reveal the role of zinc finger protein 263 in glioma EMT and further explore the underlying molecular mechanisms.MethodsTechnical methods in molecular biology,cell biology and histology were applied and divided into two parts: in vitro and in vivo.In vitro,the expression of ZNF263 in human brain glioma tissue was observed by western blot and immunohistochemistry.The relationship between the expression of ZNF263 and the overall survival of glioma patients was analyzed by online database prediction.The knockdown and overexpression cell lines of ZNF263 and Furin were then constructed by plasmid transfection,respectively.Proliferation and colony formation ability were observed by CCK-8 assay and plate colony formation assay.Migration and invasion ability of glioma cells were detected by wound healing and Transwell assay.Furthermore,to better reveal ZNF263's functionality in EMT related markers(N-cadherin,Slug,Vimentin,Twist)and molecules in TGF-? signaling pathway(TGF-?1,p-Smad2/3)were detected by western blot.Besides,the regulation of ZNF263 on Furin and the effects of Furin on TGF-?1and EMT were also observed by western blot.Gain and loss of ZNF263 and Furin in glioma cells was set up for positive and negative validation.Finally,the effects of ZNF263 and Furin on glioma EMT were verified in vivo by constructing a subcutaneous tumor-forming transplantation model in nude mice followed by immunohistochemistry.ResultsWestern blot,immunohistochemistry,and online database analysis showed that the expression of ZNF263 was significantly higher in human brain glioma tissues compared with normal control.Furthermore,the ZNF263 expression level was negatively correlated with the overall survival of patients(P<0.05).CCK-8 assay showed that decreased ZNF263 expression in glioma cells LN229 and U251 transfected ZNF263 sh RNA significantly inhibited cell proliferation(P < 0.05).Reduced colony formation was observed in LN229 and U251 ZNF263 sh RNA groups by plate colony formation assay.The subcutaneous tumorigenic transplantation model in nude mice showed that the weight and volume of subcutaneous tumors in the ZNF263 sh RNA group were smaller than those in the NC group,and the volumetric growth rate of subcutaneous tumorigenic cells was also significantly slower(P < 0.05).Proteins were extracted from LN229 and U251 cells transfected ZNF263 sh RNA or normal control.Western blot showed that the expression of EMT-related markers(N-cadherin,Slug,Vimentin,Twist)were reduced with the downregulation of ZNF263(P < 0.05).Wound healing and Transwell assays showed lower cell migration rate and less cells crossing the Transwell compartment in the ZNF263 sh RNA group(P < 0.05).While upregulated expression of EMT related markers(N-cadherin,Slug,Vimentin,Twist)was observed,wound healing and Transwell assays also showed higher migration rate and more cells crossing the Transwell compartment compared with the NC group(P < 0.05).Immunohistochemical staining also reconfirmed the lower in vivo expression of Twist in ZNF263 sh RNA tumor tissues.Mechanistically,in glioma cells LN229 and U251,Western blot showed that knockdown of ZNF263 could decrease the expression of TGF-? 1and p-Smad2/3,while overexpression of ZNF263 could upregulate the expression of TGF-?1 and p-Smad2/3.The up-regulation of TGF-?1 and p-Smad2/3 by overexpression of ZNF263 was inhibited by the application of LY2109761(dual inhibitor of T?RI and T?RII)(P < 0.05).Immunohistochemical also confirmed the decreased in vivo expression of pSmad2/3 by down-regulation of ZNF263.In addition,western blot also showed decreased Furin expression in LN229 and U251 transfected ZNF263 sh RNA,while increased Furin expression in A172 overexpression ZNF263(P<0.01).The expression of TGF-?1 and EMT markers(Ncadherin,Slug,Vimentin,Twist)was decreased in A172 and LN229 transfected Fuirn sh RNA but increased in LN229 overexpressing Furin(P < 0.05).The results of wound healing and Transwell assays were consistent with those of Western blot.The in vivo experiments also confirmed that downregulation of ZNF263 decreased the expression of Furin.Twist expression was also lower in the subcutaneous tumor inoculated with glioma cells transfected Furin sh RNA.Conclusions1.ZNF263 plays an oncogenic role in glioma.2.Reduced ZNF263 can inhibit the proliferation of glioma cells.3.ZNF263 promotes glioma EMT through activation of TGF-?/Smad signaling pathway,which in turn promotes the migration and invasion of glioma cells.4.Furin is regulated by ZNF263,promotes the production of TGF-?1and EMT of glioma. |
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