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Yuan-Jie Liu,1,2,* Jie-Pin Li,1– 3,* Shu-Hong Zeng,1,2 Mei Han,1 Shen-Lin Liu,1,2 Xi Zou1 1Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 2Department of No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, People’s Republic of China; 3Department of Oncology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, Jiangsu, 215600, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xi Zou; Shen-lin Liu Email zxvery@126.com; lsljsszyy@126.comPurpose: Gastric cancer (GC) is a common type of cancer worldwide. It can relapse and metastasize even after standard treatment; therefore, it has a poor prognosis. Moreover, sensitive biomarkers for prognosis prediction in GC are lacking. In this study, using a bioinformatics approach, we aimed to examine the value of DAZ Interacting Protein 1 (DZIP1) as a prognostic predictor and therapeutic target in GC.Methods: We explored the clinical relevance, function, and molecular role of DZIP1 in GC using MethSurv, cBioPortal, TIMER, Gene Expression Profiling Interactive Analysis, IMEx, ONCOMINE, MEXPRESS, and EWAS Atlas databases. The GSE118919 dataset was used to plot receiver operating characteristic curves. Using The Cancer Genome Atlas, we developed a Cox regression model and assessed the clinical significance of DZIPs. In addition, we used the “xCELL” algorithm to make reliable immune infiltration estimations. Western blot and immunohistochemistry were used to examine protein expression. The results were visualized with the ‘ggplot2ʹ and “circlize” packages.Results: In GC patients, DZIP1 was over-expressed at both the mRNA and protein levels. High levels of DZIP1 were found to be associated with poor survival in patients with GC. Our results indicated that DZIP1 could be involved in multiple cancer-related pathways such as the PI3K-Akt signaling pathway, WNT signaling pathway, and RAS signaling pathway, and its expression was correlated with the infiltration of activated myeloid dendritic cells, naive CD4+ T cells, and naive CD8+ T cells. Furthermore, we found that mutations in DZIP1 were correlated with a good prognosis in GC patients. Finally, we demonstrated a correlation between hypomethylation of the DZIP1 gene promoter and a poor prognosis in GC.Conclusion: This study is the first to demonstrate a significant correlation between high levels of DZIP1 and a poor prognosis in GC patients. Our results clarify multiple potential mechanisms that could contribute to this correlation and may thus provide novel insights into the clinical diagnosis and treatment of GC.Keywords: gastric cancer, expression, DZIP, methylation, mutation, epithelial–mesenchymal transition, immune infiltration

Yuan-Jie Liu,1,2 Jie-Pin Li,1– 3 Ying Zhang,1,2 Meng-Jun Nie,1,2 Yong-Hua Zhang,3 Shen-Lin Liu,1,2 Xi Zou1 1Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu, 210029, People’s Republic of China; 2No. 1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, People’s Republic of China; 3Department of Oncology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, Jiangsu, 215600, People’s Republic of ChinaCorrespondence: Shen-Lin Liu; Xi Zou Email zxvery@126.com; lsljsszyy@126.comPurpose: Follistatin-related gene 3 (FSTL3), an established oncogene, can modulate target gene expression via members of the transforming growth factor β (TGF-β) superfamily. The present study was conducted to evaluate the expression of FSTL3 in gastric cancer (GC) and to determine its prognostic significance. We also evaluated the possible mechanisms involved in the oncogenic role of FSTL3 in gastric carcinogenesis and development.Methods: We obtained data from the Human Protein Atlas, MethSurv, cBioPortal, UALCAN, TIMER, GEPIA, STRING, GeneMANIA, ONCOMINE, and MEXPRESS databases and examined it using R software. RNAi was used to establish stable FSTL3-knockdown (shFSTL3) and overexpression (OE) cell strains. Western blot; enzyme-linked immunosorbent (ELISA); and immunohistochemical (ICH), immunofluorescence, and phalloidin staining were used for examining protein expression. Cell invasion and migration were determined using transwell and scratch-wound assays. After tumor-associated macrophage (TAM) generation, co-culturing of cancer cells with TAMs was performed to confirm the relationship between FSTL3 and TAMs.Results: In GC patients, FSTL3 mRNA and protein levels were upregulated. FSTL3 expression was significantly linked to cancer stage as well as to pathological tumor grade in GC. Moreover, a high expression of FSTL3 was associated with a dismal survival duration in patients with GC. Furthermore, functional enrichment analysis demonstrated that FSTL3 overexpression could activate epithelial–mesenchymal transition (EMT) by promoting F-actin expression and BMP/SMAD signaling. Finally, immunofluorescence staining confirmed that the overexpression of FSTL3 promoted the proliferation of M2 TAMs.Conclusion: Taken together, our findings suggest that FSTL3 may be involved in GC progression via the promotion of BMP/SMAD signaling-mediated EMT and M2 macrophage activation.Keywords: FSTL3, biomarker, gastric cancer, EMT, M2 macrophages