Expression And Biological Function Of MAFG In Human Osteosarcoma

Background:Osteosarcoma(OS)is a common malignant bone tumor in children and adolescents.Treatment included surgical excision of local lesions combined with perioperative chemotherapy.With the progress of surgical technique and chemotherapy,the prognosis of patients with osteosarcoma is improved,but the survival rate of patients has not been significantly increased.This yields a 5-year event free survival(EFS)of approximately 70%for patients with localized osteosarcoma.However,the patients with metastatic or recurrent disease fare poorly with overall survival rates of less than 20%.There has been no breakthrough in more than 30 years.Therefore,with the in-depth study of the molecular mechanism of the occurrence and development of osteosarcoma,the discussion of OS biological targeted therapy which is different from traditional therapies has become a focus of clinical and basic research.Recent studies have found that MAFG expression is very low in normal human tissues,but its expression in tumor tissues is significantly enhanced.It is suggested that MAFG is another potential cancer-promoting factor.But its role in human OS is still unclear.This study is intended to detect the expression of MAFG in OS cells and human OS tissues,to explore the effect of MAFG expression on the biological functions of OS cells,and to explore the relationship between miRNA-4660 and MAFG in order to find the potential novel treatment strategy of OS.Methods:1.The real-time quantitative PCR(qPCR),Western blotting and other methods were applied to examine the expression levels of MAFG mRNA and protein in human OS tissues,adjacent tissues,and established(MG-63,U2OS)and primary human OS cells.2.Genetic methods,including shRNA-mediated gene silencing,CRISPR/Cas9-induced gene knockout and ectopic gene overexpression by viral construct were applied to alter MAFG expression in OS cells,their effects on growth,proliferation,apoptosis,migration and invasion of OS cells were evaluated.3.Genetic strategies,including ectopic overexpression by viral construct,mutation and forced inhibition,were applied to alter miRNA-4660 expression in OS cells.Their effects on MAFG expression,OS cell growth,proliferation,migration and invasion were tested.Results:1.The expression of MAFG mRNA is significantly increased in human OS tissue and OS cells,and its expression is relatively low in normal bone tissues.Western blot analysis showed that MAFG and HMOX1 proteins in OS tumor tissues were also significantly up-regulated.2.In U2OS,MG63 cell lines and primary OS cells,MAFG shRNA effectively inhibited cell growth,proliferation,migration and invasion,as well as cause apoptosis.In addition,knocking out MAFG using the CRISPR/Cas9 gene editing method also inhibited the growth,proliferation,migration and invasion of OS cells.In contrast,ectopic overexpression of MAFG in OS cells increased cell growth,proliferation,and migration.3.Bioinformatics analyses found that MAFG 3'-UTR(3'-non-coding region)binds directly to miRNA-4660;Forced overexpression of miRNA-4660 in OS cells inhibited the expression of MAFG;Conversely,inhibition of miRNA-4660 in OS cells increased MAFG expression;Ectopic overexpression of miRNA-4660 in OS cells inhibited cell proliferation,migration and invasion,and induced cell apoptosis.Conclusion:Overexpression of MAFG promotes OS cell progression.miRNA-4660 silenced MAFG,thereby inhibiting OS cell progression.