MYH9 Suppresses Autophagy And Promotes Glioma Malignant Activities Via USP14/c-Myc/p62 Axis

Objective:Glioma is a tumor originating from brain glial cells,as the world’s highest incidence and mortality of primary intracranial tumor,the current clinical treatment includes the largest surgical resection and supplemented by conventional chemoradiotherapy and targeted drug therapy,epidemiological results show that the incidence and mortality of glioma is still increasing year by year.The etiology of gliomas is complex and related to exposure to high doses of ionizing radiation,genetic mutations,diet,and infection.Although a large number of literature reports its related pathogenesis,there is still a lack of a clear explanation.MYH9 is widely present in humans,and its expression is associated with the pathogenesis of many tumors.It has been reported that MYH9 is involved in the pathogenesis of glioma,but its relationship with autophagy in glioma has not been reported.This study aims to further study the cancer-promoting mechanism of MYH9 in glioma,hoping to provide help for completing the pathogenesis of glioma and provide ideas for the diagnosis and treatment of glioma.Methods:(1)The expression of MYH9 in glioma tissue and normal brain tissue was detected by using The Cancer Genome Atlas(TCGA)database and the Genotype-Tissue Expression(GTEx)database,and the expression level of MYH9 in glioma tissue and its impact on prognosis were analyzed.The Western blot experiment measured the expression of MYH9 in normal glial cells and brain glioma cells.(2)Crispr/cas9 technology was used to create MYH9 total knock models in LN229 and U87 cell lines,and the effect of MYH9 expression on the proliferation of glioma cells was detected by in vitro functional experiments.(3)The interaction between MYH9 protein and c-Myc protein and ubiquitin-specific deubiquitinase USP14 in glioma cells was detected by Co-IP and Western blot experiments,and the location of their interaction was determined by immunofluorescence colocalization.The effect of MYH9 expression on the half-life of c-Myc protein was detected by Cycloheximide(CHX)and Proteasome Inhibitor(MG132).The effect of MYH9 expression on the interaction of c-Myc protein with ubiquitin was detected by Co-IP.(4)The effect of MYH9 expression on autophagy was detected by Western blot experiment.The effect of MYH9 expression on autophagy was further detected by LC3-GFP single-fluorescent lentiviral system and GFP-m RFP-LC3 dual-fluorescent lentiviral system.(5)The interaction between P62 protein,c-Myc protein and USP14 in brain glioma cells was detected by Co-IP and Western blot experiments,the location of their interaction was determined by immunofluorescence colocalization,and the effect of P62 expression on the interaction between c-Myc protein and ubiquitin was detected by Co-IP.(6)The effect of MYH9 expression on the USP14/c-Myc/P62 signaling axis in glioma cells was detected by Western blot experiment.Results:(1)The expression of MYH9 in glioma is higher than that in normal brain tissue,and high expression of MYH9 indicates a poor prognosis.MYH9 is expressed higher in brain glioma cell lines than in normal glial cells.(2)The high expression of MYH9 can increase the proliferation,migration,invasion and drug resistance of glioma cells.(3)MYH9 protein interacts with USP14 and c-Myc protein,and MYH9 protein can be degraded by ubiquitination of USP14 antagonistic c-Myc protein in the cytoplasm.(4)The high expression of MYH9 can inhibit autophagy in glioma cells.(5)P62 protein interacts with c-Myc protein and USP14,and c-Myc protein mediates the interaction between USP14 protein and ubiquitin in the cytoplasm,and then inhibits the degradation of P62 protein.(6)MYH9 regulates autophagy by regulating the USP14/c-Myc/P62 axis.Conclusion:In this study,by further exploring the cancer-promoting mechanism of MYH9 in glioma,we found that MYH9 as a cancer-promoting factor,could inhibit the level of autophagy in brain glioma by regulating the USP14/c-Myc/P62 signaling axis,and play a cancer-promoting role in promoting proliferation,migration,drug resistance,etc.indicating that MYH9 may become a potential therapeutic target.