KPNA2 Promotes Metabolic Reprogramming In Glioblastomas By Regulation Of C-myc

Backgroud:More and more researches have suggested that up-regulated glycolytic metabolism plays an important role in the proliferation and malignant progression of cancers.As one of the most common intracranial malignancies,gilomas are 3 times higher than normal brain tissues in glycolysis.The mechanism of glycolysis is still unclear.In our previous study,KPNA2 could promote proliferation,invasion and glycolytic metabolism in glioma cells.More over,some crucial factors such as c-myc.and E2F1 were potential cargo proteins of KPNA2.It suggested that KPNA2 may play an important role in promoting the metabolic reprogramming in gliomas by regulating these factors.In this study,by detecting the expression of KPNA2 in gliomas,and performing functional test of KPNA2 in vivo and in vitro,and verifying the interaction of key factors,we revealed the key role and potential mechanism of KPNA2 in the metabolic reprogramming in gliomas.Methods:A total of 90 glioma samples and 15 normal human brain samples were collected,including 30 astrocytomas(WHO grade ?),30 anaplastic astrocytomas(WHO grade?)and 30 glioblastomas(WHO grade ?).The expression of KPNA2 was analyzed by qRT-PCR(Quantitive real time polymerase chain reaction)and IHC(Immunohistochemistry).To investigate the role of KPNA2 in the energetic metabolism of gliomas,we separately transfected a lentiviral vector of KPNA2 shRNA,wild type KPNA2 or their comparative controls into the U87 and U251 glioblastoma cell lines.And then detected the activities of key enzymes such as HK,PKM and PFK involved in glycolytic metabolism by colorimetric method,and the glucose uptake and the production of lactic acid were measured in KPNA2-knockdown or overexpressed glioma cells.Then we detected the expression of Glutl by flow cytometry,cell proliferation by CCK8 and EdU assay,and cell migration and invasion by transwell assay.Moreover,to detect the influence of KPNA2 in vivo,we planted the KPNA2-knockdown U87 cells and the scrambled control U87 cells into the right flanks of nude mice.After that,through performing of Co-IP,subcellular fraction assay,immunofluorescence,western blot and dual luciferase reporter assay,we detected the interaction of KPNA2,c-myc and E2F1 and explored the mechanism of KPNA2 in the metabolic reprogramming.Results:1.QRT-PCR and IHC assays:KPNA2 was highly expressed in the glioma tissues compared with the normal brain tissues,and the expression of KPNA2 was increased with the WHO grades.KPNA2 is a crucial predictor of prognosis in the glioma patients.2.The function of KPNA2 in vitro:Knockdown of KPNA2 in the glioblastoma cell lines U87 and U251 decreased deoxyglucose uptake,activities of the key glycolytic enzymes and lactate production.Moreover,tumor proliferation and invasiveness were concomitantly downregulated.3.In vivo experiments:When the KPNA2-knockdown U87 cells subcutaneously implanted into the immunocompromised mice,apparent decrease in tumor formation and subsequent increase in the survival of the tumor-bearingmice were observed.In addition,glycolytic metabolism was inhibited when the expression of KPNA2 was knocked down.4.C-myc as a potential mediator of KPNA2.The expression of KPNA2 significantly changed the subcellular distribution of c-myc as well as its expression.5.E271,another key cargo protein of KPNA2,was further identified to play a potential role in regulating the transcription of c-myc by KPNA2.Conclusions:Our findings suggest that KPNA2,a potential tumor oncogene,performs its function at least in part via regulating cellular metabolism through c-myc signaling axis.It would provide a possible explanation for Warburg effect and thus offer a new perspective to the roles of KPNA2 in gliomagenesis.