LDH Isoenzymes Regulate The Role Of Mesenchymal Transition And Its Mechanismin Glioma

Objective:In this study,we tested the profile of lactate dehydrogenase(LDH)isoenzymes in glioblastoma multiforme(GBM),and the correlation with GBM subtypes was analysed.We further investigated the functions and the mechanisms of LDH isoenzymes regulating the mesenchymal transition.This study might offer an important theoretical basis for LDH isoenzymes-targeted treatment.Methods:(1)Bioinformatic analyses were used to assess the clinical significance of LDHA and LDHB in GBM;(2)Immunohistochemistry was used to analyse the expression prolife of LDHA and LDHB in GBM,and their correlation with MES markers;(3)qRT-PCR,Western blot and native-PAGE were used to determine the LDH isoenzymes in four glioma cells and investigate the correlation between LDH subunits with MES or PN markers,plus lactate production and pyruvate consumption;(4)After knocking down LDHA or LDHB,we investigated the LDH isoenzymes by native-PAGE;CCK8 and colony formation assay were used to assess the proliferation of glioma cells,also the wound healing assay and transwell migration assay were applied to assess the migration;(5)Western blot was used to analyze MES and PN markers for investigating how LDH isoenzymes shift affected mesenchymal transition in glioma cells;(6)The chemiluminescence detection system and dehydrogenase colorimetric assay were used to detect the effects of LDHA or LDHB knockdown on lactate production and pyruvate consumptionin in glioma cells.Results:(1)The prolife of LDH subunits in mesenchymal GBM was high-LDHA and low-LDHB,while in CpG island methylator phenotype proneural subtype was low-LDHA and high-LDHB;(2)LDHA expression was positive correlated with MES markers levels in GBM samples but LDHB was not apparent;(3)The prolife of LDH isoenzymes varied in 4 glioma cells.There were mainly LDH4 and LDH5 in LN229 and U87 MG cells,with high lactate production and low pyruvate consumption,while LDH1 in SW1783 and U251 MG cells with low lactate production and high pyruvate consumption.MES markers were high in LN229 and U87 MG cells,however,PN markers were high in SW1783 and U251 MG cells;(4)LDH isoenzymes shifted after knocking down LDHA or LDHB in glioma cells;LDHA knockdown decreased the proliferation and migration,while LDHB knockdown moderately enhanced the proliferation but had no effects on migration in LN229 and U87 MG cells.Whereas LDHA knockdown had no effects,LDHB knockdown remarkably inhibited the proliferation and migration in U251 MG cells;(5)LDHA underexpression extremely inhibited the mesenchymal transition,while LDHB underexpression had no effects in LN229 and U87 MG cells;LDHA underexpression had no effects on mesenchymal transition,while LDHB knockdown prevented the process in U251 MG cells;(6)LDHA knockdown inhibited the lactate production and enhanced the pyruvate consumption in glioma cells,LDHB knockdown had no effects on the lactate production and partly impacted the pyruvate consumption.Conclusions:(1)LDH4 and LDH5 were associated with MES phenotype of GBM,and LDH1 was associated with PN phenotype;(2)LDH4 and LDH5 enhanced the proliferation and migration of MES phenotype glioma cells.They also promoted the mesenchymal transition and glycolysis;(3)LDH1 increased the proliferation and migration of PN phenotype glioma cells.Moreover,LDH1 activated the mesenchymal transition and pyruvate consumption.