Molecular Mechanism Of RNA Binding Protein MOV10 Affecting Apoptosis And Autophagy Of Glioma Cells By Regulating LncRNA DLEU1

Objective:To prove whether MOV10 is a risk factor affecting the occurrence and development of glioma through bioinformatics prediction and clinical sample analysis,and to study whether MOV10 can affect the proliferation,invasion,migration,apoptosis and autophagy of glioma cells by regulating the expression of lncRNA DLEU1 at the cellular level.Methods:1.The expression level of MOV10 in pan-cancer and glioma and the prognosis of patients were analyzed by bioinformatics,and verified by clinical tissue samples and glioma cells.(1)Analysis of the expression level of MOV10 in pan-cancer in TCGA data.(2)The expression of MOV10 in gliomas was analyzed by TCGA,CGGA and GEO database.(3)The difference of MOV10 expression was analyzed with tumor pathological grade,IDH mutation and 1p/19 q chromosome co-deletion state.(4)According to the median of MOV10 expression,the survival curve of glioma patients was drawn(5)The expression level of MOV10 and clinical factors of glioma patients were analyzed for survival.(6)The expression of MOV10 in gliomas and normal brain trauma tissues was detected.(7)The expression of MOV10 in human astroglial normal cells and glioma cells was detected.2.To determine the effect of MOV10 on the biological function of glioma cells.(1)RT-q PCR and Western blot were used to determine the interference efficiency of MOV10 si RNA.(2)After silencing MOV10,the effects of MOV10 on proliferation,invasion,migration and apoptosis of glioma cells were evaluated by MTS,clone formation,wound-healing,transwell,flow cytometry and Western blot to detect related marker proteins.(3)The correlation between MOV10 and autophagy was analyzed by bioinformatics,and the autophagy level of glioma cells was evaluated after silencing MOV10.3.To determine that MOV10 binds and regulates lncRNA DLEU1.(1)Through bioinformatics prediction to find the lncRNA of MOV10 interaction.(2)RNA binding protein immunoprecipitation(RIP)assay was used to verify the binding relationship between MOV10 and lncRNA DLEU1.(3)A glioma cell line with stable and low expression of MOV10 was constructed,and then lncRNADLEU1 was overexpressed.Finally,the gene expression efficiency and the regulation of MOV10 on lncRNADLEU1 were determined by RT-q PCR.4.To determine the effect of MOV10 on the biological function of glioma cells by regulating lncRNADLEU1.(1)After lentivirus transfection,the proliferation,invasion,migration and apoptosis of glioma cells were evaluated by MTS,clone formation,wound-healing,transwell,flow cytometry and Western blot detection of related marker proteins.(2)m RFP-GFP-LC3 adenovirus was used to evaluate autophagy flux and detect LC3 and p62 proteins to evaluate the changes of autophagy level of glioma cells.Results:1.Pan-cancer analysis showed that MOV10 was highly expressed in 19 tumors,including gliomas.In TCGA,CGGA and GSE21354 data,the expression level of MOV10 in glioma tissues was significantly higher than that in corresponding normal or paracancerous tissues.The expression level of MOV10 was higher in IDH wild group and 1p/19 q non-deletion group,and increased with the increase of pathological grade of glioma,and the higher the expression level of MOV10,the shorter the survival time and the worse the prognosis.Compared with normal brain tissues,MOV10 was significantly up-regulated in glioma tissues,and MOV10 expression was significantly higher in glioma cells than in normal human astrocytes.2.After transfection of MOV10 si RNA,the expression of m RNA and protein in MOV10 was significantly down-regulated.The results of MTS,clone formation,wound-healing,transwell and the results of flow cytometry showed that after silencing MOV10,the proliferation,migration and invasion ability of glioma cells decreased significantly,the level of apoptosis increased,and the expression of CDK6,Cyclin D1,N-Cadherin,MMP-2,Bcl-2 protein decreased significantly,while the expression level of Bax increased.Through bioinformatics analysis,it was found that MOV10 was significantly correlated with LC3 and p62,and autophagy level increased,p62 protein down-regulated and LC3II/LC3 I ratio increased after silencing MOV10.3.Through bioinformatics prediction,the interaction between MOV10 and lncRNADLEU1 was found and their binding sites were obtained.RIP experiments show that MOV10 and lncRNA DLEU1 combine with each other.The results of RTq PCR showed that the glioma cell line with low expression of MOV10 was successfully constructed,silencing MOV10 could down-regulate the expression of lncRNA DLEU1,and the overexpression efficiency of lnc DLEU1 virus was higher.4.The results of clone formation,MTS,wound-healing and transwell experiments showed that the proliferation,migration and invasion of glioma cells were significantly decreased after silencing MOV10,but the overexpression of lncRNA DLEU1 could rescue these effects.The results of flow cytometry showed that overexpression of lncRNA DLEU1 could inhibit apoptosis of glioma cells.Western Blot results showed that after silencing MOV10,the expression of CDK6,Cyclin D1,N-cadherin,MMP-2and Bcl-2 protein decreased,while the expression of Bax protein increased.Overexpression of lnc DLEU1 could inhibit the changes of the above protein.After transfection with lentivirus with low expression of MOV10,p62 protein was downregulated,LC3II/LC3 I ratio and autophagy flux were increased in glioma cells,but these effects could not be reversed after overexpression of lnc DLEU1.Conclusion:This study proved that MOV10 is highly expressed in glioma patients,and its high expression is related to poor prognosis.We have confirmed that knocking down MOV10 can promote autophagy of glioma cells,and MOV10 can affect the biological functions of glioma cells such as proliferation,invasion,migration and apoptosis by regulating lncRNADLEU1.