The Mechanism Of Glucose Metabolism Regulation By Hypoxia Effector Tenascin C In Glioma Stem Cells

Purpose: Glioblastoma(GBM)is the most commonly malignant primary brain tumor with poor prognosis.Due to the existence of glioma stem cells(GSCs),GBM exhibits various pernicious biological behaviour,such as proliferation,recurrence and chemoradiotherapy resistance.Tumor hypoxic microenvironment is vital for the maintenance of glioma stem celluar homeostasis.Upon going through the stimulation of hypoxia,GSCs will utilize the glucose metabolism reprogramming to maintain proliferation.Therefore,it is appropriate for the exploration united the hypoxic microenvironment,GSCs and glucose metabolism reprogramming,and revelating the mechanism of their interaction can provide a neo-direction for the treatment of GBM.Method: Firstly,the screening of the public database used by bioinformatical tools aimed to obtain the target gene,tenascin C(TNC).Then we employed various way,such as immunohistochemistry,immunofluorescence,qRT-PCR,western blotting and chromatin immunoprecipitation(chip)ect.,to verified the correlation between TNC and GSCs and explored the specific mechanism of hypoxia regulating TNC expression in GSCs.Next,we conducted multiple experiments in vivo and vitro to investigate the self-renewal ability of GSCs by knockdown TNC.To figure out how hypoxic microenvironment involved in the glucose metabolic reprogramming of GSCs,we have performed the glucose uptake kit,pyruvate and lactic acid detection kit,western blotting,immunofluorescence.Similarly,we employed the glucose uptake kit,pyruvate and lactic acid detection kit,western blotting,immunofluorescence and protein immunocoprecipitation(Co-IP)to reflect the affecting of TNC on glucose metabolic reprogramming and reveal its specific mechanism.Finally,by applying the inhibitor CORM-2 of TNC,we conduced the evaluation of therapeutic effect and examined the restrictions on growthing of GSCs in vivo(20 mg/kg)and vitro(60 μM).Result: We found that TNC had a preferential expression in the pseudopalisade region in GBM tissue,and our data showed that TNC exhibited obvious co-localization with GSCs markers(Sox2,Olig2,CD133)and hypoxia related proteins(HIF1 α and CA9).It was found that TNC was highly expressed in GSCs cells compared with serum-induced differentiated glioma tumor cells,and the level of protein and mRNA was upregulated under hypoxic microenvironment.Further studies showed that up-regulated HIF1α can bind to HRE site2 located in the promoter region of TNC to accelerate TNC transcription under hypoxia.After knocking down the expression of TNC in GSCs cells,the spheroidizing ability,cell activity and cell proliferation were decreased,and the apoptosis related proteins cleaved caspase3 and cleaved PARP were increased.Moreover,in the mice which undergone intracranial xenotransplant interfered the expression of TNC,the volume of tumor exhibited smaller and the survival showed a significative promotion.Our experiments showed that the uptake of glucose and production of lactate were increased in hypoxic GSCs.Consistently,the expression of glycolytic pathway related proteins PDK1,HK II and LDHA were up-regulated and PDHA1 was down-regulated.We found that the elevation of PDK1 and HK II were mainly affected by HIF1α,and insterestingly,LDHA was not only regulated by HIF1α but also HIF2α.Meaningfully,after knockdown TNC,the uptake of glucose and the production of lactate were decreased,as well as the protein level of PDK1.Furthermore,we found that TNC binding to PDK1 could affect each other in hypoxic GSCs,so the inhibition of TNC will accelerate the lysosomal degradation of PDK1,resulting in the increase of ROS production.Similarly,the down-regulation of PDK1 can contribute to the deficiency of spheroidizing ability and activity.And we observed that the overexpression of TNC resulted in the increase of glucose uptake and lactate production.Next,we established the GSCs cell lines which both over-expressed PDK1 and inhibited TNC,it was found that the glucose uptake and the lactate production were increased.Upon the concentration of CORM-2reached 60 u M,the protein level of TNC immediately displayed a suppression phenomenon,and the ability and activity of spheroidization of GSC cells were decreased.Worth mentioning,applying CORM-2 to treat intracranial tumorigenic mice generated that the volume of tumor exhibited smaller and the survival showed a significative promotion.During the treatment period,TNC,PDK1 and Ki67 were decreased in xenograft tumors,but22 days after drug withdrawal,they were obviously rasied again.Conclusion: This study found that HIF1α can regulate the expression of TNC protein in hypoxic GSCs.Up-regulated TNC can bind to PDK1 to lessen the lysosomal degradation of PDK1 in the cytoplasm,which facilitate the glucose metabolism reprogramming and reduce the production of ROS in mitochondria.Therefore,GSCs show the maintaining of self-renew under hypoxia.The applying of TNC inhibitor,CORM-2,have shown a promise in the treatment of GSCs.Our work discovers an available target and provides a neo-direction for the prosperity to achieve the radical cure of GBM.