The Functional Elucidation & Mechanism Study Of HnRNPA2B1 In Gastric Chemoresistance

【Background】Gastric cancer（GC）is the fifth most common malignancy and fourth most mortal tumor in the world.In China,GC is the most common malignant tumors in the digestive system.According to a report by the National Cancer Center in 2020,the incidence and mortality of GC in China ranked third among all malignant tumors.Chemotherapy is the basic means of clinical treatment for GC.Especially for stage II-III gastric cancer,perioperative chemotherapy can significantly reduce tumor size and improve progression-free survival and overall survival of GC patients.For stage IV gastric cancer,palliative chemotherapy can lead to longer survival and better quality of life for patients.However,chemotherapy resistance is an important cause of tumor treatment failure,recurrence and metastasis.Heterogeneous nuclear ribonucleoprotein A2B1（hnRNPA2B1）is an RNA-binding protein,which is also an N6-methyladenosine（m⁶A）recognition protein involved in RNA synthesis,processing,transport,and degradation.It has been reported that the expression of hnRNPA2B1 is upregulated in a variety of tumors,and participates in malignant progression such as tumor proliferation,invasion and metastasis by affecting downstream target RNA.However,hnRNPA2B1 has been poorly studied in gastric cancer,and it is still unclear whether it is involved in chemotherapy resistance of GC.【Objectives】1.To explore the expression and prognostic value of hnRNPA2B1 in gastric cancer chemotherapy.2.To clarify the role of hnRNPA2B1 in gastric cancer chemotherapy resistance.3.To explore the mechanism of hnRNPA2B1 in gastric cancer chemotherapy resistance.【Methods】1.Bioinformatic analysis was used to explore the expression and prognostic value of hnRNPA2B1 in pan-cancer.Western blot,real-time quantitative PCR,immunofluorescence and immunohistochemical staining were used to detect the expression of hnRNPA2B1 in gastric cancer cell lines,chemotherapy resistance cells,and gastric cancer tissues.The Kaplan-Meier Plotter database was used to analyze the prognostic value of hnRNPA2B1 in gastric cancer.2.Lentiviral transfection was used to up-and down-regulate hnRNPA2B1 in GC cell lines to establish a model of hnRNPA2B1 overexpression or loss of function.The effects of hnRNPA2B1 on tumor malignant phenotypes such as proliferation ability and chemotherapy resistance were explored in vitro and in vitro by half maximal inhibitory concentration（IC50）assay,cell proliferation curve assay,flow cytometry,nude mice xenograft assay,and immunohistochemical（IHC）staining.To explore the mechanism of hnRNPA2B1 in gastric cancer chemotherapy resistance.3.Transcriptome sequencing combined with the database of CLIP-seq were used to screen out RNA which could bind to hnRNPA2B1.The RNA co-immunoprecipitation assay（RIP）was used to detect the binding capacity of hnRNPA2B1 to lnc RNA NEAT1.Real-time quantitative PCR and Western blot were applied to detect the effect of hnRNPA2B1 overexpression or knockdown on NEAT1.RNA stability assay was used to determine the effect of hnRNPA2B1 on the half-life of NEAT1 after actinomycin D treatment.The antisense oligonucleotide（ASO）vector of NEAT1 was constructed and was used to silence NEAT1 in gastric cancer cell lines.The effects of NEAT1 loss-of-function on the proliferation capacity and chemotherapy resistance were explored by IC50 assay and cell proliferation curve assay.RNAscope in situ hybridization assay was used to detect the expression and localization of hnRNPA2B1 and NEAT1 in clinical tissues of gastric cancer.4.Sphere formation was used to detect the effect of hnRNPA2B1 on the stemness of GC cell lines.Real-time quantitative PCR,Western blot,and immunofluorescence were used to detect the effect of hnRNPA2B1 and NEAT1 on the expression of caner stem cell markers.Multiplex immunohistochemical staining was used to detect the expression of CD133 and CD44 in clinical specimens of gastric cancer.【Results】1.Bioinformatic analysis showed that hnRNPA2B1 was highly expressed in a variety of tumors.Univariate COX regression and Kaplan-Meier survival analysis found that high expression of hnRNPA2B1 was associated with poor prognosis.Western blot,q PCR,and immunofluorescence verified that hnRNPA2B1 expression was elevated in a variety of gastric cancer cell lines,and that the expression levels of chemotherapy resistant GC cells were significantly higher than those in parent cells.Immunohistochemical staining in gastric cancer tissue microarray showed that the expression level of hnRNPA2B1 in GC tissues was significantly higher than that in adjacent normal tissues.Kaplan-Meier Plotter database showed that hnRNPA2B1 was strongly associated with the prognosis of gastric cancer patients and was especially associated with poor prognosis in patients receiving 5-Fluorouracil chemotherapy.2.Western blot and q PCR verified that lentiviral transfection could persistently downregulate the expression level of hnRNPA2B1 in SGC7901^ADR and SGC7901^VCR cells and upregulate the expression level of hnRNPA2B1 in SGC7901 cells.IC50 assay,cell proliferation curve assay,flow cytometry,and Western blot showed that knockdown of hnRNPA2B1 reduced proliferation,drug-resistance ability,and promoted apoptosis of SGC7901^ADR and SGC7901^VCR cells,while overexpression of hnRNPA2B1 enhanced proliferation and drug-resistance ability of SGC7901 cells and eliminated apoptosis.Subcutaneous tumorigenic experiments in nude mice showed that knocking down hnRNPA2B1 inhibited tumor growth in vivo and reduced chemotherapy resistance of gastric cancer cells.Furthermore,IHC staining showed that the proportion of Ki-67 positive cells and Cleaved Caspase-3 positive cells increased significantly.To explore the mechanism of hnRNPA2B1 in gastric cancer chemotherapy resistance.3.Transcriptome sequencing combined with POSTAR3 database implied that lnc RNA NEAT1 may bind to hnRNPA2B1.RNA Immunoprecipitation showed that hnRNPA2B1 could significantly enrich NEAT1.q PCR demonstrated that down-regulation of hnRNPA2B1 could reduce the expression of NEAT1.RNA stability assays have shown that hnRNPA2B1 increases the stability of NEAT1.q PCR verified that ASO transfection effectively downregulated the expression of NEAT1 in hnRNPA2B1-overexpressing SGC7901 cells.IC50 assay and cell proliferation curves have shown that knockdown of NEAT1 reduces the proliferative capacity and chemotherapy drug-resistance ability of gastric cancer cells.RNAscope assays were carried out in GC clinical specimens,and it was found that hnRNPA2B1 and NEAT1 were co-localized in the nucleus,and the expression of hnRNPA2B1 and NEAT1 was significantly increased in gastric cancer tissues relative to adjacent normal tissues.4.Spearman correlation analysis showed that the hnRNPA2B1 gene expression level was significantly positively correlated with tumor stemness score.Transcriptome sequencing combined with ss GSEA demonstrated that the 10 stemness-related gene signatures were enriched in the ADR＿sh NC group,indicating that knockdown of hnRNPA2B1 may impair cancer stemness in GC.The results showed that downregulation of hnRNPA2B1 inhibited the cancer stemness and the expression of cancer stemness-related markers in SGC7901^ADR and SGC7901^VCR cells,and upregulation of hnRNPA2B1 in SGC7901 and HGC27 cells promoted the cancer stemness and the expression of stemness-related markers.In hnRNPA2B1-overexpressing SGC7901 cells,silencing NEAT1 could inhibit the expression of stemness markers,moreover,knockdown of NEAT1 could also downregulate the expression of stemness markers in gastric cancer cell lines such as MGC803 and BGC823 cells.Multiplex immunohistochemical staining showed that the expression of CD133 and CD44 was significantly increased in GC tissues and was positively correlated with the expression of hnRNPA2B1.【Conclusions】1.The expression of hnRNPA2B1 in gastric cancer cells,gastric cancer tissues,and gastric cancer chemotherapy-resistant cell lines was increased,and it was associated with poor prognosis and chemotherapy resistance in gastric cancer patients.2.hnRNPA2B1 can enhance proliferation and chemotherapy-resistance ability and inhibit apoptosis of gastric cancer cells in vitro and in vivo.3.hnRNPA2B1 can directly bind with lnc RNA NEAT1 and increase its stability,leading to enhanced chemotherapy resistance in gastric cancer.4.hnRNPA2B1 may participate in chemotherapy resistance by regulating the stemness of gastric cancer cells.