RNA-binding motif protein 28 (RBM28) plays a critical regulatory role in various malignancies, yet, its expression pattern and functional role in gastric cancer remain unclear. This study aims to investigate the effect and molecular mechanism of RBM28 on the development and progression of gastric cancer (GC).

① Study materials and clinical sample grouping: Patients who visited Department of Gastroenterology of Daping Hospital between December 2021 and December 2023 and from whom tissue samples were collected in this study. Among them, 14 normal gastric mucosal tissues were assigned to Normal group, and 17 gastric cancer tissue samples were assigned to GC group. By integrating the data from The Cancer Genome Atlas (TCGA) database and our in-house RNA-seq data, differentially expressed members of the RBM family were screened, and RBM28 was identified as a significantly altered candidate gene. ② Tissue-level validation and prognostic analysis: Immunohistochemistry was performed to detect RBM28 protein expression in the Normal and GC groups. In addition, transcriptomics, and survival data of GC patients from the Kaplan-Meier Plotter database (n=876) were used to analyze the association between RBM28 expression and patient prognosis. ③ Cell model construction and functional assays: GC cell lines HGC-27 and AGS were employed as experimental models. Stable RBM28-knockdown cell lines and their corresponding control cells were established via lentiviral infection. Cell proliferation was assessed using CCK-8 assay at days 1, 2, 3, and 5 after seeding, with 6 replicate wells per group. Colony formation assay was also performed to evaluate clonogenic proliferative capacity. ④ Cell cycle analysis: Propidium iodide (PI) staining combined with flow cytometry was utilized to observe cell cycle distribution of the control and RBM28-knockdown groups, and to determine the effect of RBM28 on G1/S phase progression. ⑤ Mechanistic investigation and rescue validation: Pathway enrichment analysis was conducted to identify key genes associated with RBM28-related cell cycle and DNA replication pathways. A DONSON overexpression rescue model was further constructed, including the Control, shRBM28, and shRBM28+DONSON groups. DONSON expression was verified by RT-qPCR. Subsequently, CCK-8 assay, colony formation assay, and flow cytometry were performed to evaluate the changes in cell proliferation and cell cycle distribution after DONSON rescue, thereby determining whether RBM28 regulates G1/S phase progression and malignant proliferation of GC cells through DONSON.

① Integrated analysis of the TCGA database and our in-house dataset, as well as immunohistochemical validation, demonstrated that the protein level of RBM28 was significantly upregulated in GC tissues (P<0.0001), and high RBM28 expression was significantly associated with poor prognosis (HR=2.36, 95%CI: 1.45 to 3.85, P=0.0004). ② Knockdown of RBM28 significantly inhibited the proliferative capacity of GC cells (P<0.01). ③ Mechanistically, we found that high RBM28 expression was closely associated with the cell cycle DNA replication pathways. RBM28 knockdown induced G1 phase arrest in GC cells (P<0.05), and DONSON was identified as a key regulatory factor involved in this process. ④ Furthermore, overexpression of DONSON following RBM28 knockdown significantly reversed G1/S phase arrest and restored the suppressed proliferative capacity of GC cells (P<0.05).

RBM28 is highly expressed in GC tissues, and promotes G1/S phase progression by upregulating DONSON expression, thereby enhancing the proliferative capacity of GC cells and driving tumor progression. The RBM28/DONSON axis may serve as a potential novel target for GC molecularly targeted therapy.

To determine the expression level of TG-interacting factor 1 (TGIF1) in gastric cancer (GC) and its correlation with prognosis, and investigate the characteristics of immune microenvironment of TGIF1-overexpressing GCs and its clinical significance.

Integrated analysis was performed using transcriptomic data from The Cancer Genome Atlas (TCGA), Asian Cancer Research Group (ACRG), and our in-house GC transcriptome database. Immunohistochemistry (IHC) assay was employed to compare TGIF1 expression between GC and normal gastric mucosa tissues. Based on Kaplan-Meier Plotter online database, the correlation between TGIF1 expression and clinical prognosis was evaluated. Transcriptomic data were analyzed to identify functional enrichment features of GC with high TGIF1 expression. The GENIE3 toolkit and STRING database were utilized to predict TGIF1-regulated target genes and protein-protein interaction (PPI) networks, respectively to explore the potential immune-related signaling pathways regulated by TGIF1. Single-cell RNA sequencing (scRNA-seq) was applied to analyze the association between TGIF1 expression and tumor microenvironment (TME) disorder.

Transcriptomic analysis revealed significantly higher TGIF1 expression in GC tissues compared to normal tissues (P<0. 01). Patients with high TGIF1 expression exhibited poorer clinical prognosis (P<0. 05). IHC assay confirmed elevated TGIF1 expression in GC tissues than normal tissues (P<0. 01). GC with high TGIF1 expression was enriched in immune regulatory pathways, including immunosuppressive cytokines such as chemokine C-C motif ligand 20 (CCL20). These tumors displayed an immunosuppressive TME, characterized by abundant immunosuppressive NK cells, mast cells, regulatory T cells (Tregs), myeloid cells, and exhausted CD8⁺ T cells. Cell interaction analysis suggested that TGIF1-overexpressing GC cells may engage with Tregs via the CCL20-CCR6 axis. Co-high expression of signature gene sets from these interacting cells was associated with significantly shorter survival in the patients (P<0. 05).

TGIF1 is highly expressed in GC tissues and may serve as a biomarker for immunosuppressive TME and poor prognosis. TGIF1-overexpressing GC cells may interact with multiple immune cells, particularly Tregs, to induce an immunosuppressive microenvironment.