The Role And Mechanism Of M6A Methyltransferase KIAA1429 In Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma(DLBCL)is the most common subtype of adult non-Hodgkin’s lymphoma being highly aggressive and heterogeneous.Although novel immunotherapies represented by CD20 monoclonal antibodies have significantly improved the prognosis of DLBCL patients,more than one-third of patients are still not cured due to drug resistance to primary therapy or relapse after remission.In recent years,the use of epigenetic drugs in DLBCL patients has played a key role in improving the prognosis of this patient population,but the efficacy remains limited.Therefore,actively exploring epigenetic alterations in DLBCL and their potential as therapeutic targets may provide new insights for optimal individualized treatment of DLBCL.Epigenetic regulation is crucial for maintaining life homeostasis,mainly including DNA methylation,histone modification,chromosome remodeling and non-coding RNA regulation.N6-methyladenosine(m6A)modification is the most abundant RNA modification in eukaryotes,which is dynamically and reversibly regulated by methyltransferases,demethylases and binding proteins,regulating RNA stability,translation,splicing and export.KIAA1429 is a newly identified component of the m6A methyltransferase complex,which plays an important role in m6A modification.Recent studies have demonstrated that KIAA1429 is aberrantly expressed in a variety of human malignancies and is closely associated with the malignant phenotype of tumors and poor patient prognosis,and is considered as a novel tumor biomarker and potential therapeutic target.However,the role of KIAA1429 in DLBCL and its mechanism still need to be further investigated.This study intended to investigate the expression level and clinical significance of m6A methylation regulators,reveal the biological role and molecular mechanism of m6A methyltransferase KIAA1429 in the development of DLBCL,elucidate new mechanisms of epigenomic regulation in DLBCL,and provide new insights into the prognosis and individualized treatment of DLBCL.Part Ⅰ.Clinical Significance and Biological Function of KIAA1429 in Diffuse Large B-cell LymphomaObjective:This study aimed to investigate the expression and clinical significance of m6A methylation regulators in DLBCL,explore the clinical value and biological function of KIAA1429 in DLBCL,and reveal its role in the development of DLBCL.Materials and Methods:1.Analysis of the expression and prognostic significance of m6A methylation regulators based on the GEO database combined with bioinformatics.2.Under mechanism examination of KIAA1429 using weighted correlation network analysis(WGCNA),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis in DLBCL for potential biological functions.3.Selection of clinical cases and collection of specimens for DLBCL and reactive hyperplasia lymphoid(RHL).4.Isolation of single nucleated cells from peripheral blood specimens of healthy controls.5.Immunomagnetic bead-based sorting of CD19+B cells.6.Real-time quantitative polymerase chain reaction(RT-qPCR).7.Western blotting.8.Immunohistochemistry(IHC)and hematoxylin-eosin(HE)staining.9.Cell culture.10.Construction of KIAA1429 stable knockdown,knockout,and endogenous overexpression cell lines.11.Cell Counting Kit-8(CCK-8)to detect cell proliferation.12.Annexin V-PE/7AAD double-staining assay for detection of apoptosis.13.PI/RNase assay for cell cycle detection.14.Construction of DLBCL xenograft mouse model.15.Statistical analysis.Results:1.Differential analysis of m6A methylation regulators based on the GEO dataset GSE56315 revealed that multiple m6A methylation regulators were aberrantly expressed in DLBCL patients.Cox regression and Lasso regression were performed on the GSE117556 dataset to screen out prognostic genes and construct a prognostic model,and the OS of patients in the high and low-risk groups according to the median risk score was significantly different(p<0.0001).The ROC curve showed that the prognostic risk model possessed high sensitivity and specificity in predicting the OS of DLBCL patients.The results of univariate and multifactorial Cox regression analysis revealed that the risk model could be acted as an independent prognostic indicator for DLBCL patients(p<0.001).The results of correlation analysis indicated that m6A methylation regulators were significantly associated with clinical prognostic indicators in patients with double-hit lymphoma(DHL)(p<0.05).Among them,high expression of KIAA1429 was negatively correlated with the OS of DHL patients.2.The expression levels of KIAA1429 in CD19+B lymphocytes from DLBCL cell lines(OCI-LY1,OCI-LY3,OCI-LY8,VAL,U2932)and healthy control peripheral blood mononuclear cells were detected,and the results revealed that the mRNA and protein levels of KIAA1429 were significantly elevated in DLBCL cell lines.Immunohistochemical staining of the enrolled 60 de vo DLBCL tissue specimens and 20 reactive lymphadenitis tissue specimens(control)revealed that the expression level of KIAA1429 was significantly higher than that of the control group(p<0.0001).Statistical analysis combined with the clinical information of the included patients showed that positive KIAA1429 expression was associated with DLBCL subtype(p=0.034)and higher Ann Arbor stage(p=0.007).The results of the survival analysis indicated that patients with high KIAA1429 expression had a shorter survival time(p=0.005).3.The GEO dataset GSE117556 was analyzed by WGCNA and divided genes into different modules according to the similarity of gene expression patterns,and the genes related to KIAA1429 were selected for GO and KEGG functional enrichment analysis,which revealed that KIAA1429 was significantly enriched in cell proliferation,cell cycle,apoptosis and cancer-related pathways.OCI-LY1 and U2932 cells were stably transfected with KIAA1429 knockdown lentivirus(shKIAA1429)and negative control lentivirus,and the knockdown efficiency of KIAA1429 at the mRNA and protein levels was verified by western blotting and RT-qPCR.Knockdown of KIAA1429 resulted in a decrease in the proliferation rate of DLBCL cells(p<0.05),an increase in the apoptosis rate of cells(p<0.01),and elevated proportion of cells in the G2/M phase of the cell cycle(p<0.001).In contrast,endogenous overexpression of KIAA1429(KIAA1429 OE)promoted DLBCL cell proliferation and inhibited DLBCL cell apoptosis.4.By transfecting DLBCL cells with CRISPR-Cas9 gene editing system targeting deletion of KIAA1429(sgKIAA1429),we found that knockout of KIAA1429 significantly inhibited DLBCL cell proliferation,promoted apoptosis and induced DLBCL cell arrest in G2/M phase.In a subcutaneous xenograft mouse model,KIAA1429 knockout reduced tumor growth rate and the expression of proliferationrelated protein Ki67.Conclusions:1.Expression of m6A methylation regulators is dysregulated in DLBCL,and the prognostic model constructed based on m6A methylation regulator can accurately predict the OS of DLBCL,and its risk score can be acted as an independent prognostic indicator of DLBCL.2.m6A methyltransferase KIAA1429 expression is elevated in DLBCL,and its high expression is significantly associated with disease progression and poor prognosis.3.KIAA1429 exerts tumor-promoting effects by regulating cell proliferation,apoptosis,and cell cycle.Part Ⅱ.KIAA1429 Promotes Diffuse Large B-cell Lymphoma Growth by Mediating m6A Modification to Inhibit Hippo/YAP Signaling Pathway ActivationObjective:The aim of this study was to investigate the regulatory mechanism of KIAA1429 in DLBCL and provide a theoretical basis for the pathogenesis,risk assessment,and development of targeted intervention strategies in DLBCL.Materials and Methods:1.DLBCL cell culture2.Lentiviral vector-mediated knockdown of CHST113.Colorimetric assay to detect overall m6A methylation levels.4.RNA Sequencing(RNA-seq)to detect differentially expressed genes in shKIAA1429 and shCon groups.5.Methylated RNA Immunoprecipitation sequencing(MeRIP-seq)to detect the level of m6A methylation modification between shKIAA1429 and shCon groups.6.RT-qPCR.7.Western blotting.8.RNA immunoprecipitation(RIP)-qPCR.9.Dual luciferase reporter gene assay assay to verify the regulation of the m6A methylation modification site of CHST11 mRNA by KIAA1429.10.Actinomycin D-based RNA stability assay.11.CCK-8 assay for cell proliferation.12.Annexin V-PE/7AAD double-staining assay for detection of apoptosis.13.PI/RNase assay for cell cycle detection.14.Co-Immunoprecipitation(Co-IP).15.Immunofluorescencetechnic(IF)and confocal microscopy.16.Cytoplasmic cytosolic protein extraction.17.Construction of DLBCL xenograft mouse model.18.Statistical analysis.Results:1.The colorimetric results showed that knockdown of KIAA1429 decreased the overall m6A level of cells.CHST11 was screened as a possible target gene of KIAA1429 by RNA-seq and MeRIP-seq.Bioinformatics analysis based on the GEO database revealed a significant correlation between KIAA1429 and CHST11(p<0.0001).RIP-qPCR experiments confirmed the binding of KIAA1429 to CHST11 mRNA(p<0.001).2.Dual luciferase reporter gene detection assay showed that KIAA1429 regulated the m6A site on the 3’UTR of CHST11 mRNA.RNA stability assay indicated that KIAA1429 decreased CHST11 mRNA stability.By RT-qPCR and RIP-qPCR,the m6A reader YTHDF2 was found to regulate CHST11 expression,and YTHDF2 bound to CHST11 mRNA(p<0.01),and the amount of binding was reduced with the downregulation of KIAA1429.3.Knockdown of CHST11 promoted DLBCL cell proliferation and inhibited apoptosis in vitro;and facilitated tumor growth in vivo.Further knockdown of CHST11 in DLBCL cells with KIAA1429 knockdown revealed that CHST11 knockdown reversed the decrease in cell proliferation and increase in apoptosis and cell cycle arrest phenotype induced by KIAA1429 knockdown.4.KEGG enrichment analysis based on our RNA-seq showed that the differential genes were significantly enriched in the Hippo signaling pathway.Correlation analysis of the GSE117556 dataset revealed association between CHST11 and core components of the Hippo-YAP pathway including MOB1B,NF2,LATS2,LATS1,and MOB1A.The interaction and co-localization of endogenous CHST11 and MOB IB in DLBCL cells were verified by Co-IP and immunofluorescence confocal imaging analysis.Western blotting results revealed that knockdown of KIAA1429 increased phosphorylation of the S127 site of YAP and LATS1,decreased total protein levels of YAP in the nucleus and cytoplasm,and these effects were reversed by knockdown of CHST11.Conclusions:The above study demonstrated that KIAA1429 exerts a tumor-promoting effect by mediating m6A modification of CHST11 mRNA,recruiting YTHDF2 to bind the m6A site,inhibiting CHST11 stability and expression,interfering with the interaction between CHST11 and MOB1B,and suppressing the activation of Hippo-YAP signaling.Our study identifies new dimensions of epigenetic alterations in DLBCL and highlights the potential of KIAA1429 as a novel prognostic biomarker and therapeutic target for DLBCL progression.