MYH9-Dependent Ubiquitination Of GSK-3β Promotes Malignant Progression And Chemoresistance In Gliomas

Objective:Glioblastoma(GBM)is the most common type in glioma with highly malignance.For quite some time,it is the main treatment that surgical resection achieving maximum safe resection with postoperative radiotherapy and chemotherapy.However,the treatment does not work well,and the median survival period of patients is still less than 15 months.The intractability of the treatment,on the one hand,is related to its high degree of malignancy and feature of invasive growth,which is bound up with the Epithelial-Mesenchymal Transitions(EMT).On the other hand,as the main postoperative chemotherapy drug currently,temozolomide(TMZ),its resistance has also become one of the main factors of the recurrence of GBM.Therefore,targeting the common pathway of EMT and TMZ resistance would hopefully improve the survival time of patients.This study would explore the molecule targeting these two pathways and its specific mechanism in GBM.Methods:First of all,through bioinformatics using TCGA,CGGA and GETx databases and experiment including PCR and immunohistochemistry with glioma samples,it has been explored that the difference and regularity of MYH9 expression in different grades of glioma,as well as the relationship with prognosis.Then,with the GBM cells being well silenced in MYH9 via transfection of lentivirus,cell proliferation test(including MTT assays and EdU incorporation assays),invasion and migration test,nude mouse transplantation tumor experiment and TMZ resistance test were carried out to explore the role of MYH9 in GBM and its relationship with TMZ resistance.The mechanism of low expression of MYH9 relieving malignant progression of GBM was preliminarily investigated by Western blot.What’s more,in order to see whether the low or high expression of MYH9 could impact the mRNA and protein level in GSK-3β,the GBM cell lines with transfection of small interfering RNA/plasmid to knockdown/overexpress MYH9 has been constructed to perform experiment.Soon afterwards co-IP and cell immunofluorescence were carried out to detect the interaction between MYH9 and GSK-3β,after which the ubiquitination site in GSK-3β and the interaction site in MYH9 has been finally dug out.Last but not least,the influence of high and low expression of MYH9 on prognosis was analyzed through the follow-up data of glioma patients.Results:1.It is shown that not only compared to normal tissue MYH9 was highly expressed in glioma and was positively correlated with WHO grade,but also its high expression indicated poor prognosis.2.MYH9 do work well in the progression of GBM through experiment in vivo and vitro.Knockdown of MYH9 weakened the proliferation,migration and invasion of GBM cells,which also alleviated the TMZ resistance.3.The inhibition that the low expression of MYH9 functions on proliferation maybe due to the reduction of Cyclin D1 and CDK4.And Silencing MYH9 also inhibited the ability of migration and invasion through Wnt/β-catenin signaling pathway,which weakened TMZ resistance.4.MYH9 site 1,peptide segment from 75th to 777th,interacted with GSK-3β followed ubiquitination in K27 and K36 to participate in Wnt/β-catenin signaling pathway.5.With the analysis of follow-up information from 86 glioma patients,it was found that the high expression of MYH9 suggested a poor prognosis and could be used as an independent prognostic factor.Conclusion:MYH9 is highly expressed in glioma and indicates adverse survival time.Knockdown of MYH9 renders GSK-3β ubiquitinated and degraded,as a result of which the malignant of GBM and its resistance in TMZ could be relieved.