LncAGTR Promotes TMZ Resistance In GBM By Regulating The Hsa-miR-10401-3p/HSPA1A Axis And Aerobic Glycolysis

Background Glioblastoma multiforme(GBM)is the most common tumor of the brain,accounting for 45-50% of all primary solid brain tumors.GBM shows invasive growth,it is difficult to completely remove the tumor by surgery,and it is more prone to drug resistance and recurrence after postoperative treatment.Temozolomide(TMZ)is the first-line standard drug for GBM treatment and can improve the prognosis of GBM patients.Unfortunately,most patients will inevitably develop TMZ resistance and lead to tumor recurrence.Even with surgery combined with postoperative chemoradiotherapy,the median survival time of patients is only 15 months,and the 5-year survival rate of of patients is less than 10%.Therefore,elucidating the mechanism of TMZ resistance and discovering new therapeutic targets is of great value to improve the therapeutic effect of GBM.Long noncoding RNAs(lncRNAs)are non-coding RNAs with more than 200 nt without or with low protein-coding capacity.Numerous studies have shown that lncRNAs play crucial roles in glioma TMZ drug resistance via different ways.Lnc RNAs are associated with aerobic glycolysis,which is an important cause of tumor drug resistance.So,is lncRNA-regulated glioma TMZ resistance related to aerobic glycolysis? There are few reports in the literature.AimsIn this study,whole transcriptome sequencing(WTS)was performed using recurrent glioblastoma multiforme(r GBM)and primary glioblastoma multiforme(p GBM)tumor tissue samples,and bioinformatics analysis including differentially expressed genes,KEGG and GSEA functional enrichment and annotation,and in vitro validation were used to screen and identify key lncRNA molecules that play a crucial role in GBM recurrence.Then,the functions and mechanisms of key lncRNAs in GBM TMZ acquired drug resistance were identified through a series of in vitro and in vivo experiments to provide potential therapeutic targets for GBM treatment.Methods(1)Screening and identification of target m RNA and lncRNA molecules: In this study,WTS was performed on 3 r GBM and 3 p GBM tumor tissues,bioinformatics analysis was used to screen differentially expressed m RNAs,KEGG and GSEA were used to analyze the function of m RNAs,CGGA database was used to analyze the relationship between HSPA1 A and GBM prognosis,q RT-PCR and Western blot were used to detect the expression of HSPA1 A in r GBM to determine the key m RNA-HSPA1 A to be studied;The ce RNAs networks formed by lncRNAs with more than 9-fold differences targeting HSPA1 A,mi RNAs and HSPA1 A were constructed;The expression of lncRNAs in r GBM was evaluated by q RT-PCR assay;The correlation between lncRNAs and HSPA1 A expression was calculated by Pearson correlation analysis to identify the key lncRNA to be studied.(2)Effects of target lncRNA on HSPA1 A expression and TMZ resistance in GBM cells: GBM cells(U251Ctrl,GBM-LCtrl)were continuously stimulated by TMZ to establish TMZ-resistant GBM cell lines(U251TR,GBM-LTR);The expression of HSP1A1 A in TMZ-resistant GBM cells were measured through q RT-PCR,IF and Western blot methods,and FISH were used to detect the expression and cellular localization of lnc AGTR in TMZ-resistant GBM cells;The expression of lnc AGTR in U251 TR and GBM-LTR cells were downregulated,the expression of lnc AGTR in U251 TR and GBM-LTR cells were upregulated,and the expression of HSPA1 A was detected by Western blot,and the TMZ-resistant properties of GBM cells were detected by CCK8.(3)Effects of target lncRNAs on GBM cells proliferation and aerobic glycolysis: The expression of lnc AGTR in U251 TR and GBM-LTR cells were downregulated,the expression of lnc AGTR in U251 TR and GBM-LTR cells were upregulated,and western blot was used to detect the changes of ERK and AKT expression;The proliferation ability of GBM cells was evaluated by CCK8 and clone formation ability methods;The cycle and apoptosis of GBM cells were detected through FCM assay;Glucose detection kit was used to detect cell sugar consumption,lactate detection kit was used to detect cellular lactate production;q RT-PCR was used to detect the expression changes of key molecules(HIF-1α、HK2、LDHA、LDHB、PDK1、PKM2、GLUT1、GLUT4)of aerobic glycolysis,and Western blot was used to detect the changes of STAT3 expression.(4)Mechanisms of target lncRNA-induced changes in GBM cells function: The ce RNA(competing endogenous RNAs,ce RNA)network formed by lnc AGTR-mi RNAs-HSPA1 A was constructed;Dual luciferase and RNA immunoprecipitation(RIP)were used to identify the predicted binding sites of lnc AGTR and mi RNAs.(5)Effects of target lncRNAs on the proliferation of GBM cells in vivo: A subcutaneous tumor model of GBM cells was constructed in mice;Lnc AGTR was targeted and the tumor volume was detected to identify the proliferation of GBM cells in mice;q RT-PCR was used to detect the expression of lnc AGTR and HSPA1A;IHC was to detect the expression of ki67.Results(1)Compared with p GBM,HSPA1 A is the most up-regulated gene with the largest differential expression in r GBM.Functional analysis shows that it is related to mitogen-activated protein kinases(MAPK).11 lncRNAs with more than 9-fold differential expression can form a network of ce RNAs with HSPA1A;3 lncRNAs are highly expressed in r GBM;1 lncRNA were identified,which has the highest correlation with HSPA1 A expression,named lnc AGTR(lncRNA activated in GBM with TMZ resistance,lnc AGTR).(2)TMZ-resistant GBM cell lines were successfully constructed;HSP1A1A and lnc AGTR were highly expressed in TMZ-resistant GBM cell lines,and lnc AGTR was mainly located in the cytoplasm;Lnc AGTR could regulate the expression of HSPA1 A and is associated with TMZ-resistant properties of GBM cells.(3)The results show that upregulation of lnc AGTR can activate the ERK and AKT signaling pathways,regulate cell cycle,and promote cell proliferation;Meanwhile,upregulation of lnc AGTR can activate the STAT3 signaling pathway,increase the expression of pyruvate kinase M2(PKM2),and promote the occurrence of aerobic glycolysis in GBM cells.In contrast,knocking down the expression level of lnc AGTR in cells yielded the opposite results.(4)Lnc AGTR upregulated HSPA1 A expression through competitive binding to hsa-mi R-10401-3p.(5)Targeting inhibition of the expression of lnc AGTR can inhibit the growth of glioma in mice,and the expression levels of lnc AGTR and HSPA1 A in tumor tissue were reduced.ConclusionLncAGTR may activate the ERK,AKT and STAT3 signaling pathways by acting on the hsami R-10401-3p/HSPA1 A axis,thereby promoting cell proliferation and increasing aerobic glycolysis,resulting in GBM resistance to TMZ and relapse.Lnc AGTR has a high potential as a predictor and therapeutic target of TMZ resistance.