Epitranscriptomic regulation of the COX-2/PGE2 axis by hnRNPA2B1 promotes esophageal squamous cell carcinoma progression

Figure 4 HnRNPA2B1 stabilizes m6A-modified cyclooxygenase-2 mRNA and promotes its expression in esophageal squamous cell carcinoma. A: Western blotting analysis of cyclooxygenase-2 (COX-2) expression in paired adjacent (A) and tumor (T) esophageal squamous cell carcinoma (ESCC) tissues from three representative patients; B: Relative COX-2 mRNA expression in paired adjacent and ESCC tissues determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR); C: Western blotting analysis of COX-2 expression in Het-1A cells and ESCC cell lines (Eca109 and KYSE150); D: Relative COX-2 mRNA expression in Het-1A, Eca109, and KYSE150 cells determined by RT-qPCR; E: Western blotting analysis of COX-2 expression in Eca109 and KYSE150 cells transduced with control short hairpin RNA or hnRNPA2B1 short hairpin RNA (sh56-1); F: Relative COX-2 mRNA expression in Eca109 and KYSE150 cells after hnRNPA2B1 knockdown, as determined by RT-qPCR; G: Western blotting analysis of COX-2 expression in Eca109 and KYSE150 cells transduced with negative control or hnRNPA2B1 overexpression vector; H: Relative COX-2 mRNA expression in Eca109 and KYSE150 cells after hnRNPA2B1 overexpression, as determined by RT-qPCR; I: Representative immunohistochemistry staining of COX-2 in normal esophageal epithelium, low-grade intraepithelial neoplasia, and high-grade intraepithelial neoplasia from endoscopic submucosal dissection specimens; J: Quantification of COX-2-positive cells in normal, low-grade intraepithelial neoplasia, and high-grade intraepithelial neoplasia tissues; K: Representative immunohistochemistry staining of COX-2 in ESCC tissues and matched adjacent and distal tissues; L: Quantification of COX-2-positive cells in ESCC, adjacent, and distal tissues; M: Methylated RNA immunoprecipitation-qPCR analysis in Eca109 cells showing enrichment of COX-2 mRNA in the m6A-immunoprecipitated fraction relative to the IgG control; N: RNA immunoprecipitation-qPCR analysis in Eca109 cells showing enrichment of COX-2 mRNA in hnRNPA2B1 immunoprecipitates, with reduced enrichment after hnRNPA2B1 knockdown and increased enrichment after hnRNPA2B1 overexpression; O: Actinomycin D assay in Eca109 cells showing COX-2 mRNA decay after hnRNPA2B1 knockdown or overexpression. (Panels A, C, E, and G were derived from the same membranes as Figure 1I and K, Figure 2A and C, respectively, so that hnRNPA2B1 and COX-2 expression could be assessed in the same samples. The corresponding loading controls are shown in Figure 1I and K, Figure 2A and C, respectively.) Data are presented as mean ± SD. Statistical significance was determined by Student’s t-test for two-group comparisons or one-way ANOVA for multiple-group comparisons, as appropriate. Scale bars = 100 μm. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. COX-2: Cyclooxygenase-2; ESCC: Esophageal squamous cell carcinoma; shCON: Control short hairpin RNA; NC: Negative control; OE: Overexpression; shA2B1: HnRNPA2B1 short hairpin RNA; LGIN: Low-grade intraepithelial neoplasia; HGIN: High-grade intraepithelial neoplasia; MeRIP: Methylated RNA immunoprecipitation; RIP: RNA immunoprecipitation.