Natriuretic peptide receptor a promotes gastric malignancy through angiogenesis process

Figures

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  Fig. 1. NPRA was upregulated in GC and associated with poor prognosis. A The expression of NPRA and CD31 from GC tissues were measured by IHC. B, C The protein level of NPRA was significantly increased in GC tissues (53/86). D Online Kaplan–Meier overall survival (OS) and relapse-free survival (RFS) curves according to NPRA expression levels. ***p < 0.001.

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  Fig. 2. NPRA promoted angiogenesis by regulating VEGF signaling pathway in GC. A GSEA analysis of the relation of NPRA expression with angiogenesis based on TCGA cohort. B The cluster heat map was used to analyze gene expression levels of subsets of genes in shNPRA GC cells relative to matched shCTL GC cells. C KEGG pathway analysis of the RNA-seq results from shNPRA and the corresponding shCTL GC cells. D GSEA analysis of subsets of genes related to the VEGF signaling pathway.

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  Fig. 3. NPRA promoted GC-associated angiogenesis in vitro. A The expression of VEGF in the culture medium of MKN45 and AGS cells transfected with shNPRA and the control group (shCTL) was detected by ELISA assay. B CCK8 assay was applied to detect cell viability of HUVECs cultured with different conditioned mediums of shNPRA and shCTL GC cells. C Tube-formation assays with HUVECs were performed with the different groups of GC conditioned medium, scale bar: 100 μm. D, E Transwell migration and invasion assays with HUVECs treated with different conditioned mediums showed that knockdown of NPRA reduced the cell ability of migration invasion, scale bar: 200 μm. F, G Flow cytometry assay was used to analyze the HUVEC cell viability and cell cycle regulated by different GC cells conditioned mediums. CM: conditioned medium, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 4. Silencing NPRA could inhibit growth of the GC organoids. A Effects of NPRA knockdown on the growth of gastric organoids, scale bar: 20 μm. B, C Transwell migration and invasion assays with HUVECs treated with conditioned medium of Patiente1-shCTL, Patiente1-shNPRA, Patiente2-shCTL, and Patiente2-shNPRA GC organoids, scale bar: 200 μm. D, E Flow cytometry assay was used to analyze the HUVEC cell viability and cell cycle that regulated by different GC organoids conditioned medium. CM: conditioned medium, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 5. NPRA facilitated GC-associated angiogenesis in vivo. A Xenograft tumors in the nude mouse model was treated with shNPRA and shCTL. B CD31 expression of NPRA-silencing xenografted tumors were significantly lower than control groups, scale bar: 50 μm. C Representative images of lung metastasis and HE staining of the specimen, scale bar: 100 μm. D The VEGF detection of tumor harvested from mouse xenograft model by western blot. **p < 0.01, ***p < 0.001.

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  Fig. 6. NPRA positively regulated HIF-1α expression resulting in angiogenesis promotion. A Western blots were employed to assess the HIF-1α expression regulated by NPRA. B Tube formation assays of HUVECs treated with the GC conditioned medium of shCTL, shNPRA, and shNPRA+HIF-1α, respectively, scale bar: 100 μm. C, D Flow cytometry assay and CCK8 assay were used to analyze the HUVEC cell viability and cell proliferation that regulated by the above GC conditioned medium. E qRT-PCR detection showed that NPRA had no significant influences on HIF-1α expression on mRNA level. CM: conditioned medium, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 7. NPRA promoted tumor angiogenesis by binding and protecting HIF-1α against protein degradation. A The proteins that bound to NPRA were determined by immunoprecipitation/mass spectrum (IP/MS) analysis, which indicated that NPRA could bind to HIF-1α at high binding scores. B Co-IP experiments demonstrated that NPRA immunoprecipitated with anti- HIF-1α antibody, and HIF-1α immunoprecipitated with anti- NPRA antibody. C, D Western blot analysis of HIF-1α protein stability using cycloheximide (CHX, an inhibitor of protein synthesis) when knockdown of NPRA. E AGS cells transfected with shNPRA and shCTL were treated with MG132, HIF-1α, and NPRA protein levels were detected by western blot.

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  Fig. 8. Schematic representation of the role of NPRA on the angiogenesis in GC. In summary, NPRA promoted GC angiogenesis by activating the HIF-1α–VEGF axis. NPRA interacted with HIF-1α protein and stabilized it to prevent its ubiquitination degradation.