Research On The Mechanism Of IDH1 Mutation Related Hsa＿circ＿IQGAP1 Promoting Malignant Growth Of Glioma

Objective Based on the cancer-promoting effect of hsa＿circ＿IQGAP1 confirmed in previous experiments,it is further determined whether IDH1 mutation and its metabolites are correlated with the expression of hsa＿circ＿IQGAP1.Meanwhile,high-throughput sequencing,HTS),liquid chromatography-tandem mass spectrometry(LC-MSMS)and bioinformatics methods were used to screen the downstream molecules that hsa＿circ＿IQGAP1 might act on,and to elucidate the specific mechanism of IDH1 mutation affecting the prognosis of glioma patients by double luciferase reporting assay.In order to provide a new molecular marker for the molecular pathological diagnosis of IDH1 molecular glioma,and to find a new target for the treatment of IDH1 molecular glioma.Methods Preliminary experiments have screened and verified the cancer-promoting effect of the target molecule hsa＿circ＿IQGAP1.By expanding clinical samples,55 IDH1 wild-type glioma tissue samples and 24 IDH1 mutant glioma tissue samples were collected.Quantitative real-time PCR(QRT-PCR)was used to measure the relative expression level of target molecules in tissue samples,and the relevant information of sample patients was obtained by returning and querying medical records,and the influencing factors of survival of patients with glioma were statistically analyzed.IDH1 mutation inhibitor(AG-120)was transfected with Small interfering RNA(IDH1)by exogenous addition of H2O2,Glutathione(GSH),2-hydroxyglutaric acid(2-HG),IDH1 mutation inhibitor(AG-120).Si RNA,et al.,simulated IDH1 mutation environment,and q RT-PCR was used to detect the effect of IDH1 mutant metabolites on the expression level of target molecules,and to explore the ways in which IDH1 mutation may affect the expression level of target molecules.Nucleo-plasma separation and Fluorescence in situ hybridization(FISH)techniques were used to locate the target molecule.RNA pull down and LC-MSMS are used to obtain proteins that the target molecule may bind to.RNA Binding Protein database prediction and RNA Binding Protein Immunoprecipitation(RIP)assay were used to verify the Binding ability of the target molecule to the screened Protein.Bioinformatics was used to predict the miRNA(micro RNA),mRNA and dual-luciferase report gene that the target molecule might bind to verify the intermolecular binding ability.Small interfering Rnas of downstream target genes were constructed to knock down downstream mRNA in glioma cell lines to verify the effects of target genes on tumor cell migration,invasion,proliferation,apoptosis and tumorigenesis.After knockdown of downstream target genes,high-throughput sequencing was performed to screen the signal pathways that the target genes might be involved in.Results 1.79 clinical samples showed that the expression level of circ＿IQGAP1 in IDH1 mutant glioma was lower than that in IDH1 wild glioma,and the difference was statistically significant(P < 0.05).Cox proportional risk regression model showed that the risk of death in patients with high circ＿IQGAP1 expression was 3.333 times higher than that in patients with low circ＿IQGAP1 expression(95%CI[1.016,10.933],P < 0.05)2.In vitro experiments,exogenous addition of H2O2 can reduce the expression level of circ＿IQGAP1,and GSH can save the decreased expression level of circ＿IQGAP1.Exogenous addition of 2-Hg had no effect on the expression of circ＿IQGAP1,ag-120 could save the decreased expression of circ＿IQGAP1 caused by IDH1 knockdown.3.Circ＿IQGAP1 is expressed in both the nucleus and cytoplasm of glioma,with higher expression in the nucleus.RIP assay failed to confirm the binding of the protein to the target molecule screened by RNA pull down results.4.Double luciferase reporter assay and rescue assay confirmed that circ＿IQGAP1 could bind mir NA-1256 and mir NA-622,and mir NA-1256 and mir NA-622 could bind downstream target genes RCAN1 and RCAN2,respectively.5.Knockdown of RCAN1 and RCAN2 in glioma cell lines can inhibit the migration,invasion and proliferation of glioma cells and promote the apoptosis of glioma cells.The results of subcutaneous tumogenesis experiment in nude mice showed that knockdown of RCAN1 and RCAN2 could inhibit the malignant growth of glioma.6.TCGA database analysis showed that RCAN1 expression was higher and RCAN2 expression was lower in tumor tissues than in normal tissues(P < 0.001).The expression level of RCAN1 was positively correlated with the pathological grade,and the difference was statistically significant.The expression level of RCAN1 in IDH1 mutant glioma was lower than that in IDH1 wild glioma,and the difference was statistically significant(P < 0.001).The expression level of RCAN2 was higher than that of IDH1 wild-type glioma,the difference was statistically significant(P < 0.01).The survival curve showed that patients with high RCAN1 expression had a worse prognosis than those with low RCAN1 expression,the difference being statistically significant(P < 0.001).7.The differential Gene Ontology(GO)analysis of high-throughput sequencing results of knockdown target genes RCAN1 and RCAN2 showed that RCAN1 may affect the activity of protein phosphatase inhibitors and affect protein binding and bridging to regulate tumor development through positive regulation of TGF-βreceptors;RCAN2 may regulate tumor development by influencing intracellular calcium homeostasis,REDOX enzyme activity and receptor activator activity.Conclusions IDH1 mutation can reduce the expression of circ＿IQGAP1 in glioma,and the high expression of circ＿IQGAP1 is an independent risk factor affecting the survival of glioma patients.IDH1 mutation may decrease the expression level of target molecule through ROS accumulation and GSH reduction.Circ＿IQGAP1 regulates downstream target genes RCAN1 and RCAN2 through the sponge action of mir NA-1256 and mir NA-622 to promote the malignant growth of glioma.Circ＿IQGAP1 has the potential to be a biomarker for the diagnosis of IDH1 molecular glioma,and may be a new target for the treatment of IDH1 molecular glioma.