ALKBH5 Regulates The Expression Of CYSLTR2 Through M~6A Modification To Promote The Proliferation Of Gastric Cancer And Its Mechanism

Objective:The incidence and mortality of gastric cancer(GC)have been high,and it is urgent to conduct in-depth research on the mechanism of gastric cancer development to improve the clinical situation.N6-methyladenosine(m~6A)modification provides a new level of regulation for gene expression,and dysregulation of the overall level of m~6A modification and the expression level of its regulator is critical for cancer initiation,progression,and metastasis.Our previous study found the demethylase Alk B homolog 5(ALKBH5)affecting gastric cancer proliferation,and what is the downstream target genes of ALKBH5 mediated gastric cancer proliferation?Is it dependent on its demethylase activity?What are the specific modification sites and binding proteins?In-depth study of these problems can broaden the understanding of gastric cancer proliferation involved in m~6A modification,and provide potential early screening indicators and therapeutic drug targets for gastric cancer.Materials and Methods:Immunohistochemistry(IHC),real-time PCR(q PCR)and western blot(WB)were used to detect the expression of various genes.Methylated RNA immunoprecipitation sequencing(Me RIP-seq)and RNA sequencing(RNA-seq)were used to screen ALKBH5 downstream target genes.PCR,site-directed mutagenesis technology and homologous recombination mediated ligation were used to construct overexpression or knockdown plasmids of each gene as well as site-directed mutant plasmids.Short hairpin RNA(sh RNA)combined with lentivirus was used to screen gastric cancer cell lines with stable knockdown of endogenous gene expression.The vitality and proliferation of gastric cancer cells were detected by CCK-8 cell viability assay,Ed U cell proliferation assay and colony formation Assay.The cell cycle was detected by flow cytometry.The growth of gastric cancer in nude mice was detected by subcutaneous tumor formation experiment.RNA-binding protein immunoprecipitation(RIP),immunofluorescence(IF)and fluorescence in situ hybridization(FISH)were used to verify the binding and co-localization of ALKBH5 with its downstream target genes.Results:ALKBH5 is up-regulated in gastric cancer tissues and gastric cancer cell lines.Functionally,we verified that ALKBH5 facilitated gastric cancer cells proliferation through in vitro and in vivo assays,respectively.CYSLTR2,as a downstream target of ALKBH5,has promoted proliferation in gastric cancer.Furthermore,knockdown of ALKBH5 markedly increased m~6A modification level of cysltr2 m RNA and decreased its expression.Caused by ALKBH5 demethylase activity mutation,downregulated expression of CYSLTR2 indicated that ALKBH5 modulates CYSLTR2 expression in an m~6A-dependent manner.We also revealed that ALKBH5-mediated cysltr2 m RNA degradation relied on YTHDF2.The overexpression of ALKBH5 abolished the m~6A modification on cysltr2 m RNA,so that YTHDF2 did not bind to m~6A modification site,and the m RNA stability of CYSLTR2 was enhanced to up-regulate the expression and promote the proliferation of GC.Conclusion:Our reserach demonstrates that ALKBH5 regulates CYSLTR2 expression to promote gastric cancer proliferation in an m~6A modification-dependent manner,and the ALKBH5 removed the m~6A modification of cysltr2 m RNA and reduced the binding of YTHDF2,resulting in decreased degradation and increased expression of CYSLTR2.Those results enrich the role of m~6A regulation in tumor malignant proliferation,and represent a new insights into potential biomarkers and therapeutic targets for gastric cancer therapy.