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The function and effect on prognosis of ANXA2 in gastric cancer peritoneal metastasis patients in cold region
doi: 10.1515/fzm-2024-0024
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Abstract:
Objective Heilongjiang Province is part of the northern cold areas of China, where gastric cancer is one of the most common gastrointestinal malignancies. Peritoneal metastases (PM) are the leading cause of mortality among patients. This study conducted bioinformatics and basic research on the gene ANXA2 (Annexin A2), which may influence the prognosis of patients. Methods Genome sequencing was performed on patients from Heilongjiang to identify potential genes impacting survival time. The function of ANXA2 in gastric cancer was analyzed using multiple bioinformatics databases, focusing on its pathways and mechanisms. ANXA2-knockout gastric cancer cell lines were constructed, and in vitro assays, including CCK-8, flow cytometry, scratch, and Transwell experiments, were conducted. A nude mouse tumorigenesis model was also developed to analyze in vivo effects. Results ANXA2 was found to be expressed at higher levels in gastric cancer tissue than in normal gastric tissue, and its mRNA levels were associated with short overall survival (OS). Enrichment analysis indicated that ANXA2 is primarily localized on the cell membrane and primarily influences the PI3K-AKT signaling pathway. Cytological experiments demonstrated that knockdown of ANXA2 suppresses the growth and migration of gastric carcinoma cells, an effect that was also observed in vivo. Conclusions ANXA2 is essential for gastric cancer growth and may represent a potential risk factor affecting the survival probability of patients in cold regions. -
Key words:
- Gastric cancer /
- ANXA2 /
- prognosis survival /
- bioinformatics /
- cold region
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Figure 1. Bioinformatics results of ANXA2
(A) Changes of ANXA2 mRNA expression levels in gastric cancer and normal gastric tissues; (B) Expression levels of ANXA2 mRNA in the tissues of gastric cancer patients at different clinical stages; (C) Immunohistochemical analysis of ANXA2 protein expression in gastric cancer and normal tissues; (D) Relationship between ANXA2 mRNA levels and the survival of gastric cancer patients.
Figure 2. Enrichment results and relationship with PDL1 and ANXA2
(A-B) Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) related to ANXA2; (C) The PI3K-AKT pathway in KEGG database; (D) Protein interaction network of ANXA2; (E) Relationship between mRNA expression of ANXA2 and PDL1; (F) mRNA expression of PDL1 in gastric cancer and normal tissues. (E) Relationship between mRNA expression of ANXA2 and PDL1; (F) Expression of PDL1 mRNA in gastric cancer and normal tissues.
Figure 3. miRNAs and TF of ANXA2
(A) Predicted Wayne diagram results of miRNAs; (B) Predicted miRNA expression in gastric cancer and normal tissues and the correlation with ANXA2 expression; (C) Predicted Wayne diagram results of transcription factors; (D) Predicted transcription factor expression in gastric cancer and normal tissues and the correlation with ANXA2 expression; (E) mRNA of SMAD3 expression in relation to the prognosis of gastric cancer patients; (F) Overall relationship among various factors.
Figure 4. ANXA2 expresson levels in different cell lines
(A) ANXA2 mRNA expression levels in HGC27 cell line; (B) ANXA2 mRNA expression levels in MKN45 cell line; (C) ANXA2 protein levels in different cell lines. ****P < 0.0001, ***P < 0.001.
Figure 5. Cell proliferation and Flowcytometric results of different cell lines
(A) Cell proliferation in the HGC27 cell line; (B) Cell proliferation in the MKN45 cell line; (C) Flow cytometric analysis of cell cycle in HGC27 NC cells; (D) Flow cytometric analysis of cell cycle in ANXA2-HGC27 cells; (E) Flow cytometric analysis of cell cycle in HGC27 cell line; (F) Flow cytometric analysis of cell cycle in MKN45 NC cells; (G) Plot of flow cytometric analysis of cell cycle of ANXA2-MKN45 cells; (H) Proportion of various cell cycles in MKN45 cells. *P < 0.05, ****P < 0.0001.
Figure 6. Metastatic ability of different cell lines
(A) Images of scratch experiments (100×) for different cell lines; (B) Representative images showing the results from Transwell experiments for various cell lines as specified; (C-D) Bar graphs of statistical results from 24-hour scratch experiments; (E-F) Bar graphs of statistical results from Transwell experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 7. In vivo results of different cell lines
(A) Photographs of HGC27 cell-implanted mice and the control littermate; (B) Photographs showing the relative tumor sizes between HGC27 cell-implanted mice and the control counterparts; (C) Statistical results of tumor weight between the two groups; (D) Time-dependent changes of tumor volume between the two groups; (E) Relative protein levels in tumor tissues; (F) Luminescence images showing abdominal implantation in mice; (G) Statistical data of luminescence intensity on abdominal implantation tumors. *P < 0.05, ***P < 0.001.
Table 1. ShRNA sequence of NC and ANXA2
ShRNA Strand ShNC Top GATCCGTTCTCCGAACGTGTCACGTAATTCAAGAGATTACGTGACACGTTCGGAGAATTTTT ShNC Bottom AATTGAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGAATTACGTGACACGTTCGGAGAA ShANXA2 Top CCGGCGGGATGCTTTGAACATTGAATTCAAGAGATTCAATGTTCAAAGCATCCCGTTTTTTG ShANXA2 Bottom AATTCAAAAAACGGGATGCTTTGAACATTGAATCTCTTGAATTCAATGTTCAAAGCATCCCG 
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