ALKBH5 Mediated Long Noncodimg RNA SOX2OT Promotes SOX2 Expression And Regulates Temozolomide Resistance In Glioma

BackgroundGlioblastoma(GBM)is the most common intracranial malignant tumor.The standard treatment is surgical resection combined with postoperative concurrent radiotherapy and chemotherapy.The patient has a poor prognosis,with a median survival period of only 14 months and a high risk of postoperative recurrence.Temozolomide(TMZ),is currently the first-line chemotherapy drug of GBM,and TMZ resistance has seriously restricted the efficacy,which has become an important cause of treatment failure.Previous studies have shown that abnormally expressed LncRNAs are:involved in the regulation of tumor development and radiochemotherapy sensitivity,suggesting that LncRNAs may play an important role in GBM chemotherapy resistance.We screened out the differentially expressed LncRNA SOX2OT and its adjacent gene SOX2 through the expression profile of LncRNAs Chip and found that both of them were significantly highly expressed in TMZ-resistant cells,suggesting that both SOX2OT and SOX2 may be involved in the TMZ-resistance process and there may be a regulatory relationship with each other.Biological information prediction indicated that SOX2OT was bound,to the RNA methylation enzyme ALKBH5 and may participate in the SOX2OT regulation of SOX2 process.The hypothesis of this study was that ALKBH5 mediates SOX2OT/SOX2 pathway to regulate TMZ resistance.in glioma.ObjectiveThis study aimed to clarify the involvement of long non-coding RNA SOX2OT in TMZ resistance process and its clinical significance,and to explore the potential mechanism of m6A demethylase alkbh5-mediated SOX2OT in the regulation of SOX2 expression in TMZ resistance.MethodsThe expression level of LncRNA or mRNA was detected by qRT-PCR.CCK8 method and flow cytometry were used to detect the sensitivity of TMZ-resistant cell lines to TMZ and the changes of cell proliferation and apoptosis after SOX2OT or SOX2 knockdown.FISH assay was used to analyze the localization of SOX2OT in cells,and RNA Pulldown and RIP assays were used to verify the binding of SOX2OT and ALKBH5.The promoter region binding of ALKBH5 and SOX2 and the modification level of m6A in the methylated region were analyzed respectively by CHIP,EMSA and MeRIP assays.M6A modification of SOX2 in resistant cells after silencing ALKBH5 was detected by m6A quantitation assay.Results1?LncRNA SOX2OT was significantly overexpressed in TMZ-resistant cells,and its abnormal overexpression was positively correlated with TMZ chemotherapy resistance and glioma WHO grade,indicating poor prognosis.2.?Knockdown of SOX2OT can significantly enhance the sensitivity of TMZ-resistant cells to TMZ,inhibit cell proliferation and increase TMZ-induced apoptosis.Overexpression of LncRNA SOX2OT can increase the chemotherapeutic resistance of parental cells,and further tumor formation in nude mice confirmed that LncRNA SOX2OT knockdown reduced the drug resistance of TMZ-resistant cells.3?The adjacent gene SOX2 is significantly highly expressed in resistant cells,and its expression level is positively correlated with the expression level of SOX2OT.Knockdown of SOX2 can significantly enhance the sensitivity of TMZ-resistant cells to chemotherapy,inhibit cell proliferation,and increase TMZ-induced apoptosis.Overexpression SOX2 can activate the Wnt/?-cantenin signaling pathway,inhibit cell apoptosis,and promote cell proliferation.4.?Cell function recovery experiment confirmed that SOX2OT promoted chemotherapy resistance through forward regulation of SOX2,and co-transfection of overexpressed SOX2 could reverse the chemotherapy sensitivity of TMZ after SOX2OT knockdown.5?The FISH assay showed that LcRNA SOX2OT was abundant in both cytoplasm and nucleus.Bioinformatics predicted that SOX2OT could bind with ALKBH5,and the RNA Pulldown and RIP experiments confirmed the interaction between ALKBH5 and SOX2OT.6?The results of ChIP and EMSA confirmed that the combination of ALKBH5 and SOX2 promoter region promoted the transcription expression of SOX2.Meanwhile,MeRIP results showed that ALKBH5 catalyzed the m6A methylation modification of SOX2.m6A quantitative results showed that the m6A modification level of TMZ-resistant cells was significantly increased after the silencing of ALKBH5,and the expression level of SOX2 was increased.Conclusion1?The up-regulation of Long non-coding RNA SOX2OT expression promotes TMZ resistance and indicates poor prognosis in glioma.2.?SOX2OT can reduce the sensitivity to TMZ in GBM cells,promote the proliferation and inhibit apoptosis of glioma cells.3.?SOX2OT regulates the sensitivity to TMZ chemotherapy through the activation of Wnt/?-catenin signal pathway mediated by SOX2.4?RNA demethylase ALKBH5 mediated SOX2OT regulation of SOX2/Wnt signaling pathway to promote TMZ-resistance in glioma.