ALKBH5-PYCR2 Positive Feedback Loop Promotes Proneural-mesenchymal Transition In GBM

Objective: This study aimed to investigate the mechanism of Alpha-ketoglutaratedependent dioxygenase and Alk B homolog 5(ALKBH5)and pyrroline-5-carboxylate reductase 2(PYCR2)in the proneural-mesenchymal transition in glioblastoma multiforme(GBM).Methods:(1)Analysis of the relationship between ALKBH5 and PYCR2 expression in glioma tissues and clinicopathological classification and prognosis by The Human Protein Atlas and Chinese Glioma Genome Altas;(2)q RT-PCR and Western blot were used to detect the m RNA and protein difference of ALKBH5 and PYCR2 in GBM cell lines,and silencing ALKBH5 and PYCR2 in relatively high expressing GBM cells,while overexpressing both genes in relatively low expressing GBM cells;(3)The ability of proliferation,migration and invasion before and after ALKBH5 and PYCR2 interference or overexpression in GBM cells were detected via cloning assay,CCK-8 assay and Transwell assay;(4)Western blot and q RT-PCR were used to detect the regulatory relationship between ALKBH5 and PYCR2 and the changes of proline content in GBM cells were detected by proline kit;(5)Western blot was used to detect the expression of proteins related to AMPK/mTOR signaling pathway;(6)Expression of ALKBH5 and PYCR2 as markers of proneural-mesenchymal transition by Western blot;(7)In vivo experiments demonstrate the effect of the ALKBH5-PYCR2 loop on tumor growth in mice.Results:(1)Clinical database results show that the expression levels of ALKBH5 and PYCR2 in GBM increase with increasing pathological grade and negatively correlate with overall survival in GBM patients;(2)Western blot results showed that ALKBH5 and PYCR2 were inconsistently expressed in the 4 GBM cells,with relatively high expression in U251 MG and U87 MG cells and relatively low expression in SW1783 and LN229 cells;(3)Results of cellular functional assays showing that ALKBH5 and PYCR2 promote proliferation,migration and invasion of human glioma cells;(4)q RT-PCR and Western blot results indicated that ALKBH5 promoted the expression of PYCR2 at the m RNA and protein levels,and that PYCR2 affects proline expression;(5)Western blot showed that inhibition of mTOR protein expression significantly reduced ALKBH5 expression in protein level,while overexpression of PYCR2 could not rescue ALKBH5 protein expression;(6)Western blot showed that ALKBH5 and PYCR2 promoted the expression of the mesenchymal phenotypic markers N-cadherin,vimentin,and YKL-40 protein and inhibited the expression of the pro-neural phenotypic marker OLIG2 protein;(7)In vivo experiments showed that ALKBH5 and PYCR2 promoted the growth of malignant gliomas in mice and that proline partially rescued the growth of gliomas,which was not significantly rescued by the addition of rapamycin.Conclusions: ALKBH5 upregulates the expression of PYCR2,which in turn promotes ALKBH5 expression via the AMPK/mTOR signaling pathway and proline synthesis,in addition forming an ALKBH5-PYCR2 positive feedback loop that promotes GBM cell proliferation,migration,invasion and proneural-mesenchymal transition.