The Clinical Value And Mechanism Of M6A Methylation Regulators In Esophageal And Ovarian Cancer

BackgroundN6-methyladenosine(m6A)modification is the most abundant epigenetic modification in eukaryotic mRNA,and it is a dynamic and reversible process.It has been found that m6A modification may play an oncogenic or tumor-suppressive role in different tumors.In view of that research direction of this subject and the result of the preliminary experiment,it is helpful to explore the biological function of m6A regulatory gene in esophageal and ovarian cancer and identify the target gene modified by m6A,which is helpful to further explore the pathogenesis of tumor and develop new therapeutic methods.Esophageal cancer(ESCA)is one of the most common and refractory malignant tumors in the world.As a high incidence area of esophageal cancer,China has the highest proportion of new cases in the world.Therefore,it is necessary to explore the pathogenesis of esophageal cancer in detail so as to provide theoretical basis for the pathogenesis and treatment of esophageal cancer.Ovarian cancer(OC)is characterized by strong invasiveness,difficult early diagnosis and easy recurrence.Based on bioinformatics analysis such as database and some experiments,our research group previously explored that the key enzyme regulating m6A modification in ovarian cancer is ALKBH5.In the second part of this study,the role of ALKBH5 in regulating m6A modification in ovarian cancer was further analyzed from clinical samples and in vitro cell level,and the target genes of m6A modification were identified by high-throughput sequencing,so as to improve the mechanism of ALKBH5mediated m6A modification in ovarian cancer.MethodsThis study is divided into two parts.In the first part,we systematically analyzed the expression and correlation of 25 m6A regulatory factors in esophageal carcinoma from TCGA database.The molecular typing and prognostic scoring models of esophageal cancer patients were constructed by consensus clustering method and LASSO algorithm,and the potential regulatory pathways and target genes of key regulatory factor HNRNPA2B1 in esophageal cancer were screened by analyzing sequencing data.The expression of HNRNPA2B1 and potential target miRNAs in esophageal cancer tissues and peripheral blood were detected by immunohistochemistry and fluorescence quantitative PCR.The interaction and regulation mechanism of HNRNPA2B1 on target miRNAs were explored by RNA immunoprecipitation,western blot and cell proliferation assay.In the second part,we detected ALKBH5 protein expression and m6A levels by immumohistochemistry,and analyzed their correlation with clinical features and prognosis of patients with ovarian cancer.Cell proliferation,cell cycle,plate cloning and cell migration assays were used to detect the phenotypic changes of ovarian cancer cells with and without ALKBH5 knockdown.The ALKBH5 knockdown ovarian cancer cells were subjected to m6A RNA methylation sequencing and transcriptome sequencing to analyze the key target genes and pathways of m6A modification mediated by ALKBH5,and the interaction between ALKBH5 and m6A modification target genes was verified by fluorescence quantitative PCR and RNA immunoprecipitation.ResultsIn the first part of this study,we found that most of the 25 m6A regulators were significantly overexpressed and positively correlated in ESCA.Prognostic scoring models were developed based on HNRNPA2B1,ALKBH5,and HNRNPG expression levels,where risk scores were strongly correlated with tumor size and clinical outcome in ESCA patients.The risk score may be an independent prognostic factor for ESCA.The high expression of HNRNPA2B1 in ESCA patients is associated with poor prognosis,distant metastasis and lymph node staging.Knockdown of HNRNPA2B1 in esophageal cancer cells decreased the expression levels of miR-17,miR-18a,miR-20a,miR-93 and miR-106b,and inhibited ESCA.cell proliferation.In the second part,we found that ALKBH5 levels were elevated in ovarian cancer patients,while overall levels of m6A modifications were reduced.ALKBH5 knockdown or knockout inhibits ovarian cancer cell proliferation,cell cycle progression and migration.In addition,ALKBH5-regulated m6A RNA modification mainly affects RNA splicing function in ovarian cancer cells.SRSF10 is a key target gene involved in alternative splicing regulation through ALKBH5-m6A.ALKBH5 knockdown resulted in increased retention of SRSF10 exon 5 and decreased expression of transcript SRSF10-211.ConclusionsThe prognostic score model constructed by ALKBH5,HNRNPG and HNRNPA2B1 can be used to predict the 3-year survival rate of esophageal cancer patients.Overexpression of HNRNPA2B1 in esophageal squamous cell carcinoma suggests poor overall survival.HNRNPA2B1 affects the prognosis of esophageal cancer by regulating the expression of miR-17-92 cluster.HNRNPA2B1 target gene miR-17 was significantly increased in peripheral blood of patients with esophageal squamous cell carcinoma,and was associated with tumor invasion depth and lymph node metastasis.ALKBH5 was highly expressed in ovarian cancer tissues,and m6A was generally expressed at a low level,which was negatively correlated with the clinical characteristics and prognosis of ovarian cancer patients.In ovarian cancer cell line A2780,m6A-modified genes were mainly enriched in alternative splicing pathways.ALKBH5-m6A regulates exon skipping events in SRSF10 exon 5 in ovarian cancer cells.