N6-methyladenosine reader hnRNPA2B1 recognizes and stabilizes NEAT1 to confer chemoresistance in gastric cancer

Abstract

Background: Chemoresistance is a major cause of treatment failure in gastric cancer (GC). Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is an N6-methyladenosine (m⁶A)-binding protein involved in a variety of cancers. However, whether m⁶A 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, immunofluorescence, 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 immunoprecipitation, 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 tissues, 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 inducing apoptosis both in vitro and in vivo. Mechanically, hnRNPA2B1 interacted with and stabilized long noncoding RNA NEAT1 in an m⁶A-dependent manner. Furthermore, hnRNPA2B1 and NEAT1 worked together to enhance the stemness properties of GC cells via Wnt/β-catenin signaling pathway. In clinical 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 stabilizes lncRNA NEAT1, which contribute to the maintenance of stemness property via Wnt/β-catenin pathway and exacerbate chemoresistance in GC.

Keywords: NEAT1; chemoresistance; gastric cancer; hnRNPA2B1; stemness.

FIGURE 1

hnRNPA2B1 was highly expressed in GC and was associated with poor clinical outcomes. (A) mRNA expression of hnRNPA2B1 in the UALCAN database. (B) Kaplan‐Meier analyses of correlations between hnRNPA2B1 expression and OS, FPS, and PPS of GC patients and patients who received 5‐Fu treatment. (C‐D) Western blotting (C), qPCR (C) and representative IF images (D) of hnRNPA2B1 in GC cell lines. Scale bars, 10 µm. (E) Representative IHC staining of hnRNPA2B1 in matched GC and adjacent normal tissues. Scale bars, 400 µm (upper) and 50 µm (lower). (F) The IHC scores of hnRNPA2B1 in matched GC and adjacent normal tissues are shown. (G) The expression of hnRNPA2B1 in 75 paired GC and adjacent normal tissues. (H) Log‐rank test for overall survival of GC patients (n = 102). GC, gastric cancer; 5‐Fu, 5‐fluorouracil; IHC, immunohistochemistry. The data are presented as the mean ± SEM. The P value was calculated by paired t test (C), Wilcoxon's matched‐pairs signed‐rank test (F),  χ² test (G) and log‐rank test (H). *P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. Abbreviations: 5‐Fu, 5‐fluorouracil; GC, gastric cancer; hnRNPA2B1, heterogeneous nuclear ribonucleoprotein A2B1; HR, hazard ratio; IHC, immunohistochemistry.

FIGURE 2

hnRNPA2B1 induced chemoresistance by promoting proliferation and inhibiting apoptosis in vitro. (A) Western blotting and qPCR were used to detect the knockdown efficiency of hnRNPA2B1 in SGC7901^VCR cells. (B‐D) IC50 values (B) and apoptosis (C and D) of SGC7901^VCR shNC and SGC7901^VCR shA2B1 cells treated with VCR and 5‐Fu. (E) Growth curves of SGC7901^VCR shNC and SGC7901^VCR shA2B1 cells treated without or with chemotherapy. (F) Western blotting and qPCR were used to detect the overexpression efficiency of hnRNPA2B1 in SGC7901 cells. (G‐I) IC50 values (G) and apoptosis (H and I) of SGC7901 lv‐NC and SGC7901 lv‐A2B1 cells treated with ADR, VCR and 5‐Fu. (J) Growth curves of SGC7901 lv‐NC and SGC7901 lv‐A2B1 cells treated without or with chemotherapy. The data are presented as the mean ± SEM. ** P < 0.01, *** P < 0.001 by one‐way ANOVA test (A), paired t test (D, F and I) and one‐way ANOVA with Dunnett's multiple‐comparison test (B, E, G and J). Abbreviations: hnRNPA2B1, heterogeneous nuclear ribonucleoprotein A2B1; IC50, half‐maximal inhibitory concentration; lv‐NC, empty overexpression control; lv‐A2B1, overexpression of hnRNPA2B1; shNC, negative control short hairpin RNA; shA2B1, short hairpin RNA of hnRNPA2B1.

FIGURE 3

hnRNPA2B1 contributed to the chemoresistance of GC cells in vivo. (A) SGC7901^VCR shNC and SGC7901^VCR shA2B1 cells were subcutaneously implanted into nude mice, which were then treated with 5‐Fu or saline (20 mg/kg, i.p. injection). The tumor volumes and tumor weight were monitored, and growth curves were plotted every 3 days. (B) Ki‐67 and cleaved Caspase3 staining and percentages in the subcutaneous tumors of SGC7901^VCR shNC and SGC7901^VCR shA2B1 cells. (C) SGC7901 lv‐NC and SGC7901 lv‐A2B1 cells were subcutaneously implanted into nude mice, which were then treated with 5‐Fu or saline (20 mg/kg, i.p. injection). The tumor volumes and tumor weight were monitored, and growth curves were plotted every 3 days. (D) Ki‐67 and cleaved Caspase3 staining and percentages in the subcutaneous tumors of SGC7901 lv‐NC and SGC7901 lv‐A2B1 cells. The data are presented as the mean ± SEM, *P < 0.05, **P < 0.01, *** P < 0.001, ns, not significant by repeated‐measures ANOVA (A and C) or Student's t test (B and D). Scale bars, 50 µm. Abbreviations: hnRNPA2B1, heterogeneous nuclear ribonucleoprotein A2B1; i.p., intraperitoneal; lv‐NC, empty overexpression control; lv‐A2B1, overexpression of hnRNPA2B1; shA2B1, short hairpin RNA of hnRNPA2B1; shNC, negative control short hairpin RNA.

FIGURE 4

hnRNPA2B1 interacted with and stabilized NEAT1 in an m⁶A‐dependent manner. (A) Heatmap of differentially expressed lncRNAs (ǀlog₂FCǀ > 1, P < 0.05, FPKM average > 0.5) in SGC7901^ADR cells after hnRNPA2B1 knockdown. Red shades represent ADR_shNC, and blue shades represent ADR_sh. (B) Venn diagram of hnRNPA2B1‐binding lncRNAs. (C) hnRNPA2B1 knockdown or overexpression resulted in corresponding changes in NEAT1 levels. (D) qPCR analysis of NEAT1 expression in SGC7901 lv‐A2B1 and SGC7901^VCR cells after METTL3 knockdown.(E) Analysis of MeRIP assays detecting NEAT1 retrieved by an m⁶A antibody in METTL3‐silenced SGC7901 lv‐A2B1 and SGC7901^VCR cells. (F) Assessment of RIP assays detecting NEAT1 retrieved by an hnRNPA2B1 antibody or by IgG in SGC7901 lv‐A2B1 and SGC7901^VCR cells. (G) Assessment of RIP assays detecting NEAT1 retrieved by an hnRNPA2B1 antibody or by IgG in METTL3‐silenced SGC7901 lv‐A2B1 and SGC7901^VCR cells.(H) Assessment of the NEAT1 half‑life (t_1/2) in hnRNPA2B1‑silenced SGC7901^ADR cells. (I) Assessment of the NEAT1 half‑life (t_1/2) in hnRNPA2B1‑silenced SGC7901^VCR cells. (J‐K) IC50 values and growth curves of SGC7901 lv‐A2B1 cells transfected with ASO‐NEAT1 and treated with chemotherapy. The P value was calculated by one‐way ANOVA test (C left, D, H, and I), paired t test (C right and E, F, and G), one‐way ANOVA with Dunnett's multiple‐comparison test (H) and two‐way ANOVA with Dunnett's multiple‐comparison test (J and K). *P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. Abbreviations: ADR_sh, hnRNPA2B1‐knockdown SGC7901ADR cell; ADR_shNC, negative control SGC7901ADR cell; ASO‐NC, negative control of antisense oligonucleotide; ASO‐NEAT1, antisense oligonucleotide of NEAT1; CRNDE, colorectal neoplasia differentially expressed; hnRNPA2B1, heterogeneous nuclear ribonucleoprotein A2B1; IgG, immunoglobulin G; IP, immunoprecipitation; lncRNA, long noncoding RNA; METTL3, methyltransferase 3; lv‐A2B1, overexpression of hnRNPA2B1; lv‐NC, empty overexpression control; MALAT1, metastasis associated lung adenocarcinoma transcript 1; NEAT1, nuclear enriched abundant transcript 1; shNC, negative control short hairpin RNA; shA2B1, short hairpin RNA of hnRNPA2B1.

FIGURE 5

hnRNPA2B1 promoted cancer stemness‐like properties in vitro. (A) The expression of hnRNPA2B1 was correlated with mRNAsi scores in the TCGA GC dataset. (B‐C) qPCR and IF illustrated that regulation of hnRNPA2B1 resulted in corresponding changes in the expression of GCSC surface markers and pluripotent transcription factors in SGC7901^ADR, SGC7901^VCR, and parental SGC7901 cells. *P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. Abbreviations: CD133, cluster of differentiation 133; CD44, cluster of differentiation 44; EpCAM, epithelial cell adhesion molecule; GCSC, gastric cancer stem cell; hnRNPA2B1, heterogeneous nuclear ribonucleoprotein A2B1; lv‐NC, empty overexpression control; lv‐A2B1, overexpression of hnRNPA2B1; LGR5, leucine‐rich repeat‐containing G‐protein coupled receptor 5; Nanog, homeobox protein Nanog; Oct4, octamer‐binding protein 4; shA2B1, short hairpin RNA of hnRNPA2B1; shNC, negative control short hairpin RNA; Sox2, SRY‐box transcription factor 2.

FIGURE 6

hnRNPA2B1 increased the self‐renewal and tumorigenic capacities of GCSCs in vitro. (A‐B) Representative images (A) and quantification (B) of the sphere‐forming abilities of SGC7901^ADR, SGC7901^VCR and SGC7901 at the indicated passages. Scale bar, 200 µm. (C) Representative images of CD133 /hnRNPA2B1 (left) and CD44/hnRNPA2B1 (right) immunostaining in SGC7901^ADR, SGC7901^VCR and SGC7901 tumor spheroids. Scale bars, 50 µm. The P value was calculated by one‐way ANOVA test (B left and middle) and paired t test (B right). *P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: A2B1, heterogeneous nuclear ribonucleoprotein A2B1; CD133, cluster of differentiation 133; CD44, cluster of differentiation 44; GCSC, gastric cancer stem cell; lv‐NC, empty overexpression control; lv‐A2B1, overexpression of hnRNPA2B1; shNC, negative control short hairpin RNA; shA2B1, short hairpin RNA of hnRNPA2B1.

FIGURE 7

hnRNPA2B1 and NEAT1 enhanced stemness and promoted chemoresistance via Wnt/β‐catenin pathway in GC. (A) GSEA analysis between hnRNPA2B1^high NEAT1^high group (n = 105) and hnRNPA2B1^low NEAT1^low group (n = 104). (B) Immunoblots of active β‐catenin, β‐catenin, c‐Myc, and cyclin D1 in hnRNPA2B1‐silenced SGC7901^ADR cells, hnRNPA2B1‐silenced SGC7901^VCR cells and hnRNPA2B1‐overexpressed SGC7901 cells cell lines respectively. (C‐D) IC50 values and growth curves of SGC7901^ADR cells after the indicated treatment (ICG‐001, 25 µmol/L for 24 h). (E‐F) IC50 values and growth curves of SGC7901^VCR cells after the indicated treatment (ICG‐001, 25 µmol/L for 24 h). (G) Immunoblots of active β‐catenin, β‐catenin, CD133 and CD44 in SGC7901^VCR cells after the indicated treatment (ICG‐001, 25 µmol/L for 24 h). (H‐I) Representative images (H) and quantification (I) of the sphere‐forming abilities of SGC7901^VCR cells after the indicated treatment. Scale bar, 200 µm. (J) Immunoblots of active β‐catenin, β‐catenin, CD133 and CD44 in SGC7901 cells after the indicated treatment (Wnt3A, 100 ng/mL for 24 h). (K‐L) Representative images (K) and quantification (L) of the sphere‐forming abilities of SGC7901 cells after the indicated treatment. Scale bar, 200 µm. The P value was calculated by two‐way ANOVA test (C‐F), and paired t test (I and L). *P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: CD133, cluster of differentiation 133; CD44, cluster of differentiation 44; FDR, false discovery rate; GSEA, gene set enrichment analysis; hnRNPA2B1, heterogeneous nuclear ribonucleoprotein A2B1; lv‐NC, empty overexpression control; lv‐A2B1, overexpression of hnRNPA2B1; NES, normalized enrichment score; P.adj, adjust P value; shNC, negative control short hairpin RNA; shA2B1, short hairpin RNA of hnRNPA2B1.

FIGURE 8

Coordinated expression of hnRNPA2B1, NEAT1, CD133 and CD44 in GC specimens. (A) Representative images of RNAscope ISH for NEAT1 in GC specimens (left panel). Scale bars: 20 µm. Multispectral staining and imaging for hnRNPA2B1, CD133 and CD44 in serial sections of GC tissues (right panel). Scale bars: 20 µm. (B) Semi‐quantification of the expression of NEAT1, hnRNPA2B1, CD133, and CD44 in chemo‐sensitive and chemo‐resistant GC tissues. *P < 0.05, ** P < 0.01, *** P < 0.001. (C) Association between hnRNPA2B1 levels and NEAT1, CD133 or CD44 levels in GC tissues. Spearman's rank correlation coefficients (r) and P values are shown. The P value was calculated by unpaired t test. Abbreviations: CD133, cluster of differentiation 133; CD44, cluster of differentiation 44; hnRNPA2B1, heterogeneous nuclear ribonucleoprotein A2B1; NEAT1, nuclear enriched abundant transcript 1.

FIGURE 9

Proposed model for the effect of hnRNPA2B1‐mediated regulation of NEAT1 expression on GC chemoresistance. hnRNPA2B1 interacts with and stabilizes lncRNA NEAT1 in an m⁶A‐dependent manner. hnRNPA2B1 and NEAT1 collectively maintain cancer cell stemness property via Wnt/β‐catenin pathway and exacerbate chemoresistance in GC. Abbreviations: CD133, cluster of differentiation 133; CD44, cluster of differentiation 44; hnRNPA2B1, heterogeneous nuclear ribonucleoprotein A2B1; m⁶A, N6‐methyladenosine; NEAT1, nuclear enriched abundant transcript 1.