The Role And Mechanism Of ALKBH5 In Regulating CCR5 Expression In Glioma In An M6A-Dependent Manner

Chapter 1 Sequencing analysis showing the m6A methylation involved in regulating CCR5 expression in gliomaObjective:Tumor microenvironment(TME)plays an important role in glioma progression.At present,the role and mechanism of m6A modification in the regulation of tumor microenvironment in glioma is still unclear.The purposes of this study are to investigate whether m6A modification is involved in regulating the tumor microenvironment of glioma,and to further explore the relevant regulatory roles and mechanisms.Materials and Methods:We collected 4 pairs of surgically resected gliomas and peri-tumor tissues from the Department of Neurosurgery,West China Hospital of Sichuan University,for m6A sequencing(m6A-seq)analysis.TRIzol method is used to extract total RNA of tissue samples,and the quantity and purity of RNA are controlled.The original RNA library is divided into two parts.One is the Input library.The other is used to capture the required mRNA with m6A antibody.After the further screening and purification,the IP library is constructed.Double-end sequencing is performed using Illumina NovaseqTM 6000 platform(LC Bio,Hangzhou,China),and the sequencing mode is set as PE150.The sequencing files are analyzed by biological information,and the final results are obtained through visualization analysis.Results:m6A-seq analysis shows that the m6A enzymes in glioma are diversely changed compared with the peri-tumor tissues.For example,ALKBH5 and METTL3 are relatively highly expressed.The number of m6A Peaks in tumor group are more than that in peri-tumor group.The sites of m6A modification are mainly distributed in the protein coding region(CDS),followed by 3 ’UTR and 5’ UTR.GO and KEGG analyses of m6A modification-related differentially expressed genes(DEGs)show that they are related to post-transcriptional modification,signal transduction,and multiple oncogenic signaling pathways.A total of 1045 DEGs are screened out,in which 294 genes are up-regulated and 751 genes are down-regulated.Through the combined analysis of m6A-seq and RNA-seq,a total of 28 DEGs related to m6A modification are identified.Among them,CCR5,which is closely related to the TME of glioma,is found to be highly expressed in glioma,while m6A modification is at a low level.Comprehensive analyses suggest that the m6A demethylases may be involved in regulating the CCR5 expression in glioma.Conclusions:1.In this study,m6A-seq analysis shows that m6A modification is involved in regulating the expression of CCR5 in glioma,thereby regulating the TME of glioma.2.The m6A demethylases may be involved in regulating the CCR5 expression.Chapter 2 Clinical and prognostic significance of ALKBH5 and CCR5 in gliomaObjective:To analyse the type of m6A demethylases that may be involved in regulating CCR5 expression,and further analyse their clinical and prognostic significance in glioma.Materials and Methods:Firstly,the expressions of ALKBH5,FTO,and CCR5 in glioma are analyzed through TCGA and CGGA databases,and the correlations between demethylases and CCR5 gene expression are analyzed combined with m6A-seq results.The prognostic significance of ALKBH5 and CCR5 in glioma is further analyzed through TCGA and CGGA databases,as well as their relationships with tissue types,tumor grade,IDH mutation,lp/19q deletion status and disease progression status.R software(v4.0.3)is used for statistical analysis.Results:Firstly,the expressions of ALKBH5,FTO and CCR5 in glioma are analyzed through TCGA database.The results show that,compared with normal brain tissues,the expressions of ALKBH5,FTO and CCR5 are all highly expressed in glioma.In addition,the expressions of ALKBH5 and CCR5 in GBM are higher than that in LGG,while the expression of FTO in GBM is lower than that in LGG.Moreover,the correlation analyses of gene expression through TCGA and CGGA databases show that ALKBH5 is positively correlated with CCR5 at the gene expression level,whereas FTO is negatively correlated with CCR5.These mean that,the expression of CCR5 may be regulated by the demethylase ALKBH5,rather than FTO.Further analysis shows that the patients with high ALKBH5 and CCR5 expression,median OS and PFS are shorter than those with low expression,which are significantly associated with poor prognosis.Patients with high expression of ALKBH5 and CCR5 are also related to the higher tumor grade,IDH wild type,lp/19q non-codel,and disease progression.The expression,prognosis and clinical correlation of ALKBH5 and CCR5 in glioma are consistent.Conclusions:1.The expression of CCR5 may be regulated by the demethylase ALKBH5,rather than FTO.2.High expression of ALKBH5 and CCR5 are also related to the higher tumor grade,IDH wild-type,1p/19q non-codel and disease progression,and are significantly associated with poor prognosis.3.The expression,prognosis and clinical correlation of ALKBH5 and CCR5 in glioma are consistent.Chapter 3 The role and mechanism of ALKBH5 in regulating CCR5 expression in gliomaObjective:Further experiments are conducted to investigate whether ALKBH5 is involved in regulating the expression of CCR5 in glioma progression and its related regulatory role.Materials and Methods:Human glioblastoma cell lines U87 and U118 are used.The stable cell lines with ALKBH5 knock-down are constructed and screened by using shRNA interference lentivirus system.CCK-8 is used to detect glioma cell proliferation,wound healing assay and Transwell are used to detect cell migration and invasion,and flow cytometry is used to detect cell apoptosis.RT-qPCR and Western blot are used to detect the expression of ALKBH5 and CCR5,as well as the related signaling pathways.The EpiQuikTM m6A RNA Methylation Quantitative Kit(colorimetric method)and the MeRIP-m6A-qPCR assay are used to detect changes in m6A modification at the global and single-gene levels.Results:The stably transfected glioma cell lines are successfully constructed by shRNA interference with lentivirus.The experimental results show that ALKBH5 knockdown could inhibit the proliferation,migration and invasion of glioma cells,and meanwhile increase the apoptosis process.In addition,ALKBH5 knock-down significantly inhibits the gene expression and protein level of CCR5,and also inhibits the activation of the PI3K-Akt/Wnt-catenin signaling pathways.Further mechanism research shows that ALKBH5 knock-down in glioma cells can increase the m6A modification levels at both the whole level and single gene CCR5,and the differences are statistically significant.Conclusions:1.ALKBH5 knock-down can significantly inhibit the proliferation,migration and invasion of glioma cells,and increase the process of apoptosis.2.ALKBH5 knock-down also can significantly inhibit the gene expression and protein translation level of CCR5 in glioma.3.ALKBH5 knock-down can increase the m6A modification at both the whole level and single gene CCR5,thus confirming that ALKBH5 can regulate the CCR5 expression in glioma TME in an m6A-dependent manner.