NFIC Promotes Glioblastoma Cell Migration And Invasion Under Hypoxia Microenvironment

Glioma is the most common primary tumor in the central nervous system.The incidence is about 6.6 cases per 100 thousand people every year.The survival rate of the most malignant glioblastoma is only 12 to 17 months,which is mostly due to the aggressive growing of glioblastoma.Even extended-range resection cannot achieve excision at cell level.In our prior research,we found that there were tumor cells within the margin between the T2/FLAIR enhancement area and normal area.What’s more,we not only found them with low proliferation and high invasion,which is different from the core of the tumor,but also found differences in protein expression by proteomic analysis,the transition between these two group of cells remains to be elucidated,though.Hypoxia microenvironment is one of the most important features of malignant solid tumors such as glioblastoma.In the hypoxia microenvironment,the invasion,angiogenesis and chemoradiotherapy-resistance of glioblastoma are promoted by the key transcription factor--HIF-1α.Previous study revealed that hypoxia occurred within the margin of the tumor.Therefore,our research focused on whether hypoxia microenvironment is an important force that facilitates the transition of glioblastoma cells.NFIC is a member of the nuclear factor family.Previous studies have suggested that NFIC regulates the differentiation of tooth germ cells through EMT pathway and regulates the migration ability of breast cancer cells through the EMT pathway.However,the relationship between NFIC and glioblastoma under hypoxia microenvironment has not been studied yet.The main content of this study is to preliminarily explore the role of NFIC in glioblastoma.Based on this,we discuss how NFIC regulates the function and specific molecular mechanism of glioblastoma cells through affecting the ubiquitination of HIF-1α under hypoxia microenvironment.This provides a new mechanism for us to explore the regulation of malignant phenotype of glioblastoma under hypoxia microenvironment and provides a new target for glioblastoma targeted therapy.Methods(1)The expression of NFIC and its prognostic value in gliomaThe expression of NFIC in gliomas and its effect on the prognosis of NFIC are analysed by using TCGA,GEO and CGGA database,and confirming this result with Western blot at protein level.(2)The function and molecular mechanisms of NFIC in gliomaWe established glioblastoma cell line with stable knockdown of NFIC,and examined the proliferation rate by CCK8 assay,migration ability by Transwell cell migration assay,and invasion with Boyden cell invasion assay,respectively.Western blot was used to confirm the change of EMT related proteins.(3)NFIC interacted with HIF-1aImmunoprecipitation combined with mass spectrometry were used to identify the proteins directly interacted with NFIC.The results were then confirmed with CoIP,immunofluorescence and expression-correlation analysis.(4)NFIC regulates the expression of HIF-1α through the ubiquitin-proteasome pathwayWestern blot was used to determine the expression of HIF-1α of glioblastoma cells transfected with NC or LV-shNFIC,respectively,under hypoxia.At the same time,we treated the cells with MG132 or DMSO,respectively,and then repeated the Western blot.CoIP was used to determine the ubiquitination level of HIF-1α in glioblastoma cells knockdown of NFIC or not.(5)Inhibition of NFIC can reverse the promotion of malignant phenotype by overexpression of HIF1A in glioblastoma cellsWe used CCK8 assay,Transwel assay and Boyden assay to confirm the proliferation rate,migration ability and invasion in glioblastoma cells overexpressed HIF1A,respectively.Using intracranial xenograft model and IHC,we futher identified the role of NFIC in glioma.Results(1)The expression of NFIC and its prognostic value in gliomaAnalysis of the expression of NFIC in gliomas and its effect on the prognosis of NFIC using TCGA,GEO,CGGA database and Western blot revealed that NFIC was highly expressed in gliomas,especially in glioblastoma,and can be used as a molecular marker for the diagnosis of GBM.Moreover,the high expression of NFIC is associated with poor prognosis in patients with glioma.(2)The function and molecular mechanisms of NFIC in glioblastomaKnockdown of NFIC significantly inhibited proliferation,migration and invasion in glioblastoma cells.With Western Blot,we also found that NFIC could promote the EMT process of glioblastoma cells.(3)NFIC interacted with HIF-1αIP combined with mass spectrometry identified that NFIC directly interacted with HIF-1α.This result was confirmed with CoIP,immunofluorescence and expression-correlation analysis.(4)NFIC regulates the expression of HIF-1α through the ubiquitin-proteasome pathwayWith Western blot,we found that inhibition of NFIC in glioblastoma cells led to decreased expression of HIF-1α under hypoxia,and it could be reversed with MG 132 treatment.What’s more,CoIP confirmed that inhibition of NFIC could increase the ubiquitination level of HIF-1α in glioblastoma cells.(5)Inhibition of NFIC can reverse the promotion of malignant phenotype by overexpression of HIF1A in glioblastoma cellsWe found that inhibition of NFIC could reversed the promotion of migration ability and invasion in glioblastoma cells overexpressed HIF1A,respectively.Using intracranial xenograft model and IHC,we identified inhibition of NFIC could reverse the malignant phenotype promoted by overexpression of HIF1A in glioblastoma cells in vivo.ConclusionThis study not only illustrates the expression of NFIC in glioblastoma and its role in the regulation of proliferation,migration and invasion of glioblastoma cells,but also provide a new mechanism for the regulation of hypoxia microenvironment of glioblastoma cells,as well as a probable mechanism underlying the transition of glioblastoma cells and a new therapy target.