The Mechanism Study Of FTO Regulating Pri-miRNA To Suppress The Progression Of Glioblastoma

Background and ObjectivesGlioma is the most common primary malignant brain tumour of the central nervous system.GBM(Glioblastoma Multiforme),WHO Ⅳ grade glioma,remains almost invariably fatal due to its high growth rate and aggressive nature.In general,the treatment of GBM consists of operation,chemotherapy and radiotherapy.Exploring other unidentified molecular modulators could contribute to our understanding of GBM malignancy and therapy resistance to provide new insights into possible treatments.As one of the most common post-transcriptional modifications of mRNAs and noncoding RNAs,N6-methyladenosine(m6A)modification regulates almost every aspect of RNA metabolism,such as mRNA splicing,stability,translation,and microRNA(miRNA)maturation,and its function is mainly determined by m6A regulators named methyltransferase complexes("writers"),demethylases("erasers")and RNA-binding proteins("readers").Fat mass and obesity-associated protein(FTO),a member of the Fe(Ⅱ)-and α-ketoglutarate-dependent AlkB family,is reported to be the first identified m6A "eraser",and plays various roles in tumorigenesis in some tumours.The specialized and systematic mechanisms of the m6A modification regulated by FTO remains largely unknown in GBM.The part Ⅰ of this paper explores the expression characteristics of FTO within glioma tissues at various WHO grades and elucidates the role played by FTO for the malignant phenotype of glioblastoma cells.The part II explores the specific process of FTO acting on the maturation of pri-miR-10a,the primary transcription product of miR-10a-5p.The third part reveals the expression characteristics of transcription factor SPI1,which regulates FTO expression,at various grades of glioma tissues and elucidates the potential of application of the SPI1-specific inhibitor DB2313 for glioblastoma treatment.Materials and methods(1)Investigating the expression characteristics of FTO in glioma tissues by the following methodsThe expression characteristics of FTO in surgically resected tissues from glioma patients of different grades were tested by immunohistochemistry and Western Blot.Explore the expression of FTO within public databases by bioinformatics analysis,and analyse the relationship between FTO expression and clinical characteristics of patients.(2)Using in vitro and in vivo experiments to explore the effect of FTO on the malignant behavior of glioma cells.Glioblastoma cell lines were infected with lentivirus overexpressing or knockdown FTO,and tumor cell lines with stable overexpression or knockdown FTO were constructed.The effect of FTO on the proliferation ability of glioma cells was determined by CCK-8 and EdU assays,and the effect of FTO on cell invasion and migration ability was investigated by Transwell assays.The tumor animal model was constructed by in situ tumorigenesis surgery in nude mice.The size of intracranial tumor growth in nude mice was detected by fluorescence imaging.The growth status of nude mice was assessed by recording the survival period.(3)Measurement of miRNA expression by RT-qPCRComparing the sequencing data of miRNA of glioma tissues between miRNA affected by m6A and HNRNPA2B1 to find the differentially expressed miRNA.The effect of FTO on the expression of pri-miR-10a and mature miR-10a-5p was investigated by RT-qPCR.(4)Exploring protein interactions by immunoprecipitationExplore the effect of FTO overexpression or knockdown on the interaction between HNRNPA2B1 and DGCR8 by Co-IP assay.Determine the m6A methylation site on pri-miR10a by MeRIP assay.(5)Construction of plasmids containing mutant site sequencesReplace "GAACU" with "GATCU" on wild-type pri-miR-10a,integrate it into the luciferase reporter gene plasmid.Perform dual-luciferase reporter assay to investigate the effect of HNRNPA2B1 on the processing of pri-miR-10a.(6)Using following methods to predict the transcription factor regulating FTO and explore its functions.The hTFtarget database was used to predict the transcription factors regulating FTO expression,and the TCGA,CGGA databases were used to analyse their expression characteristics for screening.Use the Jaspar database to predict the sites where the FTO promoter region binds to transcription factors.Transfect the small interferences of SPI1 to glioma cell lines and detect the changes in related protein indicators by Western blot assay.The binding of transcription factors to the FTO promoter region was determined by CHIP assay.Mutant sequences of the promoter region were designed and dual luciferase reporter assay experiments were performed to determine the regulatory relationship between SPI1 and the promoter region of FTO.(7)explore the oncogenic effect of DB2313 after subcutaneous tumor injection in nude miceU87MG cell line was used for subcutaneous tumor injection in nude mice.Nude mice were divided into drug administration group and control group,and DB2313 and control solvent were injected intraperitoneally.Then,observe tumor growth size and perform statistical analysis.After that,tumor tissues were taken,fixed and sectioned.Then,to assess the expression of each protein,immunofluorescence staining was performed.Results(1)Low FTO expression suggests poor clinical prognosis and FTO inhibited the proliferation,invasion and migration of glioma cell linesBy analyzing the FTO expression data from tumor databases TCGA and CGGA the result was found that the expression of FTO decreased with glioma grade increasing.The expression of FTO was lower in mesenchymal glioblastoma than in proneuronal subtype and classical subtype.Patients with low FTO expression had shorter survival.Immunohistochemistry and Western blot experiments of clinical samples showed consistent results.The lentiviruses infection leads to the overexpression or knockdown of FTO in tumor cell lines.Then,CCK-8,EdU,Transwell and 3D tumor sphere invasion assays revealed that FTO could decrease the ability of glioblastoma cells of invasion to adjacent normal brain tissues.Intracranial GBM cells injection experiments in nude mice suggested that overexpression of FTO inhibited the growth of glioma cell lines,as well as their invasion into adjacent normal brain tissue.Glioma cells with knockdown of FTO had stronger proliferation and invasion abilities compared to control group cells.(2)The processing of Pri-miR-10a is regulated by m6A modification.FTO inhibits the production of miR-10a-5pRT-qPCR assay showed that overexpression of FTO was able to suppress the expression of mature miR-10a-5p and increase the expression of pri-miR-10a.The RIP assay showed that HNRNPA2B1 can interact with pri-miR-10a,the precursor of miR-10a-5p.Co-IP assay of HNRNPA2B1 suggest that it binds to DGCR8,which is consistent with the previous study,and FTO overexpression was able to inhibit the interaction.The interaction was not diminished after the digestion of RNAase,indicating that its binding was not RNA dependently.Site-mutated dual-luciferase reporter assay experiments suggested that mutations resulted in the inhibition of pri-miR-10a maturation.MTMR3 is a target of miR-10a-5p.Western blot assays indicated that the tranfection of miR-10a-5p mimics was able to rescue the upregulation of MTMR3 protein levels caused by FTO overexpression,and the changes in other protein levels were also rescued by miR-10a-5p mimics.(3)SPI1 promotes the proliferation,invasion and migration of glioma cell lines by inhibiting FTO expressionThe hTFtarget database predicted that SPI1,a transcription factor regulating FTO transcription,has a high enrichment score in brain tissue.The JASPAR database predicts that SPI1 binds to the FTO promoter region "GGAA" locus.After knockdown of SPI1 using small interferences,Transwell and CCK-8 experiments showed that the proliferation,invasion and migration of U118MG and U87MG were inhibited.This effect was rescued by co-transfection of FTO small interferences.Also,PCNA and Vimentin protein expression levels were reduced.CHIP experiments showed that SPI1 was able to bind to the FTO promoter region.The luciferase reporter assay experiments showed that knocking down SPI1 resulted in relatively higher firefly fluorescence values produced by the dual luciferase reporter gene plasmid of the FTO promoter,suggesting that SPI1 interacts with the FTO promoter region and represses its transcription.Western blot assays showed that U118MG and U87MG cell lines treated with DB2313 showed increased expression of FTO and MTMR3 and decreased expression of CD44 and PCNA.CCK-8 and Transwell assays showed that DB2313 inhibited the malignent behavior of glioma cells.After subcutaneous injection of tumour cells in nude mice,DB2313 was administered intraperitoneally and tumor growth was inhibited in the treated group compared to the control group.Conclusions(1)The expression of FTO decreased with glioma grade increasing.Low expression of FTO was associated with poorer clinical prognosis,suggesting the potential of FTO serving as a molecular biomarker for determining the prognosis of glioma patients.FTO inhibits cell proliferation,invasion and migration in glioblastoma cell lines.(2)The process of pri-miR-10a is regulated by the m6A mechanism.The m6A reader HNRNPA2B1 is involved in this process,which subsequently promotes the malignant progression of glioblastoma.FTO inhibits the maturation of miR-10a-5p.FTO inhibits the proliferation,invasion and migration of glioblastoma by miR-10a-5p/MTMR3 pathway.(3)SPI1 inhibits the transcription of FTO by directly binding to the FTO promoter region,resulting in its low expression.SPI1-targeted drug DB2313 could reverse the low expression of FTO and thus inhibit the malignant progression of glioma.