Background:Chemoresistance is a major cause of treatment failure in gas-tric cancer(GC).Heterogeneous nuclear ribonucleoprotein A2B1(hnRNPA2B1)is an N6-methyladenosine(m^(6)A)-binding protein involved in a variety of ...Background:Chemoresistance is a major cause of treatment failure in gas-tric cancer(GC).Heterogeneous nuclear ribonucleoprotein A2B1(hnRNPA2B1)is an N6-methyladenosine(m^(6)A)-binding protein involved in a variety of can-cers.However,whether m^(6)A modification and hnRNPA2B1 play a role in GC chemoresistance is largely unknown.In this study,we aimed to investigate the role of hnRNPA2B1 and the downstream mechanism in GC chemoresistance.Methods:The expression of hnRNPA2B1 among public datasets were analyzed and validated by quantitative PCR(qPCR),Western blotting,immunoflu-orescence,and immunohistochemical staining.The biological functions of hnRNPA2B1 in GC chemoresistance were investigated both in vitro and in vivo.RNA sequencing,methylated RNA immunoprecipitation,RNA immunoprecipi-tation,and RNA stability assay were performed to assess the association between hnRNPA2B1 and the binding RNA.The role of hnRNPA2B1 in maintenance of GC stemness was evaluated by bioinformatic analysis,qPCR,Western blotting,immunofluorescence,and sphere formation assays.The expression patterns of hnRNPA2B1 and downstream regulators in GC specimens from patients who received adjuvant chemotherapy were analyzed by RNAscope and multiplex immunohistochemistry.Results:Elevated expression of hnRNPA2B1 was found in GC cells and tis-sues,especially in multidrug-resistant(MDR)GC cell lines.The expression of hnRNPA2B1 was associated with poor outcomes of GC patients,especially in those who received 5-fluorouracil treatment.Silencing hnRNPA2B1 effectively sensitized GC cells to chemotherapy by inhibiting cell proliferation and induc-ing apoptosis both in vitro and in vivo.Mechanically,hnRNPA2B1 interacted with and stabilized long noncoding RNA NEAT1 in an m^(6)A-dependent man-ner.Furthermore,hnRNPA2B1 and NEAT1 worked together to enhance the stemness properties of GC cells via Wnt/β-catenin signaling pathway.In clin-ical specimens from GC patients subjected to chemotherapy,the expression levels of hnRNPA2B1,NEAT1,CD133,and CD44 were markedly elevated in non-responders compared with responders.Conclusion:Our findings indicated that hnRNPA2B1 interacts with and stabi-lizes lncRNA NEAT1,which contribute to the maintenance of stemness property via Wnt/β-catenin pathway and exacerbate chemoresistance in GC.展开更多
基金National Natural Science Foundation of China,Grant/Award Numbers:82073197,82273142,82222058。
文摘Background:Chemoresistance is a major cause of treatment failure in gas-tric cancer(GC).Heterogeneous nuclear ribonucleoprotein A2B1(hnRNPA2B1)is an N6-methyladenosine(m^(6)A)-binding protein involved in a variety of can-cers.However,whether m^(6)A modification and hnRNPA2B1 play a role in GC chemoresistance is largely unknown.In this study,we aimed to investigate the role of hnRNPA2B1 and the downstream mechanism in GC chemoresistance.Methods:The expression of hnRNPA2B1 among public datasets were analyzed and validated by quantitative PCR(qPCR),Western blotting,immunoflu-orescence,and immunohistochemical staining.The biological functions of hnRNPA2B1 in GC chemoresistance were investigated both in vitro and in vivo.RNA sequencing,methylated RNA immunoprecipitation,RNA immunoprecipi-tation,and RNA stability assay were performed to assess the association between hnRNPA2B1 and the binding RNA.The role of hnRNPA2B1 in maintenance of GC stemness was evaluated by bioinformatic analysis,qPCR,Western blotting,immunofluorescence,and sphere formation assays.The expression patterns of hnRNPA2B1 and downstream regulators in GC specimens from patients who received adjuvant chemotherapy were analyzed by RNAscope and multiplex immunohistochemistry.Results:Elevated expression of hnRNPA2B1 was found in GC cells and tis-sues,especially in multidrug-resistant(MDR)GC cell lines.The expression of hnRNPA2B1 was associated with poor outcomes of GC patients,especially in those who received 5-fluorouracil treatment.Silencing hnRNPA2B1 effectively sensitized GC cells to chemotherapy by inhibiting cell proliferation and induc-ing apoptosis both in vitro and in vivo.Mechanically,hnRNPA2B1 interacted with and stabilized long noncoding RNA NEAT1 in an m^(6)A-dependent man-ner.Furthermore,hnRNPA2B1 and NEAT1 worked together to enhance the stemness properties of GC cells via Wnt/β-catenin signaling pathway.In clin-ical specimens from GC patients subjected to chemotherapy,the expression levels of hnRNPA2B1,NEAT1,CD133,and CD44 were markedly elevated in non-responders compared with responders.Conclusion:Our findings indicated that hnRNPA2B1 interacts with and stabi-lizes lncRNA NEAT1,which contribute to the maintenance of stemness property via Wnt/β-catenin pathway and exacerbate chemoresistance in GC.
