TFF3 initiates gastric intestinal metaplasia by activating JAK2 and STAT3 under high salt conditions

Abstract

The mechanism underlying the role of trefoil factor family 3 (TFF3) in intestinal metaplasia remains unclear. This study reveals the molecular mechanism by which TFF3, in the process of gastric mucosal epithelial cell intestinal metaplasia (IM) induced by high salt, activates the JAK2/STAT3/CDX2 pathway, providing a potential target for the occurrence of IM. An in vitro model of high salt-induced intestinal metaplasia was established using bioinformatics to screen the GEO dataset for significantly differentially expressed genes related to intestinal metaplasia. The gastric epithelial cell line GES-1 was cultured in high-salt medium, and changes in cell function and the expression of TFF3, JAK2, STAT3, and CDX2 were examined following TFF3 knockdown or overexpression. Subsequent experiments disrupted the TFF3-JAK2/STAT3-CDX2 pathway to assess its effects on gene expression and cell function. The expression of TFF3 is upregulated during intestinal metaplasia, which promotes cell proliferation and migration. TFF3 regulates the expression of JAK2, STAT3, and CDX2 and activates the JAK2/STAT3 pathway to induce CDX2 expression in gastric epithelial cells, leading to intestinal metaplasia. Functional assays revealed that the TFF3-JAK2/STAT3-CDX2 pathway enhances both cell proliferation and migration. TFF3 induces intestinal metaplasia in gastric epithelial cells through the JAK2/STAT3-CDX2 pathway, providing new insights into the underlying mechanism and therapeutic strategies for intestinal metaplasia.

Keywords: CDX2; High salt; Intestinal metaplasia; JAK2/STAT3; TFF3.

Fig. 1

Construction of the gastric mucosal epithelial cell intestinalization model. (A) Expression levels of CDX2, VIL1, KLF4, and MUC2 after 24 h treatment with different NaCl concentrations (20, 40, 80, and 100 mM) in GES-1 cells; (B) Expression levels after treatment with 80 mM NaCl for varying durations (4 h, 8 h, 12 h, 24 h, and 48 h); (C) Expression levels after 24 h treatment with different concentrations of CDCA (50, 62.5, 75, 87.5, and 100 µM); (D) Expression levels after treatment with 100 µM CDCA for varying durations (4 h, 8 h, 12 h, and 24 h). CDCA Chenodeoxycholic acid, NC Negative control, ns not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2

Differential gene expression in intestinalization (A) Heatmap of differential gene expression; (B) Volcano plot of upregulated and downregulated genes in intestinalization; (C) LASSO regression for differential gene selection; (D) SVM regression for differential gene selection; (E) Venn diagram of the seven differential genes common to both LASSO and SVM-RFE in intestinalization; (F,G) qRT-PCR detection of TFF3 (F) and CCL25 (G) expression levels in gastric epithelial and high-salt-induced intestinalized cells. **P < 0.01, ****P < 0.001.

Fig. 3

TFF3 expression in gastric tissues. (A) Representative immunohistochemical images of TFF3 in normal and intestinal metaplasia tissues. Scale bar: 50 μm; (B) Quantitative analysis of representative images; (C) Quantitative analysis of TFF3 immunohistochemistry results in 15 cases of normal and intestinal metaplasia. ***P < 0.001, ****P < 0.0001.

Fig. 4

Impact of NaCl and TFF3 on cell proliferation and migration. (A) Line graph of CCK8 results; (B,C) Analysis and group difference statistics of the effects of high-salt and overexpression or knockdown of TFF3 on GES-1 cell proliferation; (D) Wound healing assay for cell migration; (E,F) Ratios of migration were calculated. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5

Impact of TFF3 on Expression of JAK2, STAT3, and CDX2. A Effects of high-salt on TFF3, JAK2, STAT3, and CDX2; B-E Knockdown and overexpression of TFF3 in GES-1 cells modulates expression of JAK2, STAT3, and CDX2. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6

siRNA Knockdown Efficiency Screening. Screening for the most efficient siRNA knockdown of JAK2 (A), STAT3 (B), and CDX2 (C) in GES-1 cells.

Fig. 7

TFF3/JAK2 perturbation impacts pathway genes. (A–D) Impact of high-salt, TFF3 overexpression, and JAK2 knockdown on TFF3, JAK2, STAT3, and CDX2 expression in GES-1 cells. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8

TFF3/STAT3 perturbation impacts pathway genes. (A–D) The effect of high-salt, TFF3 overexpression, and STAT3 knockdown on TFF3, JAK2, STAT3, and CDX2 expression in GES-1 cells. ns: not significant, ***P < 0.001, ****P < 0.0001.

Fig. 9

TFF3/CDX2 Perturbation Impacts Pathway Genes. (A–D) High-salt, TFF3 overexpression, and CDX2 knockdown effects on TFF3, JAK2, STAT3, and CDX2 expression in GES-1 cells. ns: not significant, ****P < 0.0001.

Fig. 10

Protein expression. (A) Validation of the TFF3-JAK2/STAT3-CDX2 signaling pathway with TFF3 overexpression and JAK2/STAT3/CDX2 knockdown. (B) Validation of the TFF3-JAK2/STAT3-CDX2 signaling pathway with TFF3 knockdown and JAK2/STAT3/CDX2 overexpression.

Fig. 11

TFF3 regulates the JAK2/STAT3 pathway via IL-6. (A) IL-6 expression levels in GES-1 cells treated with NaCl and TFF3, determined by ELISA; (B) Levels of JAK2, p-JAK2, STAT3, p-STAT3, and CDX2 in GES-1 cells treated with 100 ng/mL and 200 ng/mL of IL-6, analyzed by Western blot; (C) Quantification of western blot results, normalized to GAPDH; (D) Schematic of the TFF3-JAK2/STAT3-CDX2 signaling pathway.

Fig. 12

Cell proliferation. (A–D) Effects of high salt, TFF3 overexpression, and JAK2/STAT3/CDX2 knockdown on GES-1 cell proliferation. ***P < 0.001, ****P < 0.0001.

Fig. 13

Cell migration. Impact of high salt, TFF3 overexpression, and JAK2/STAT3/CDX2 knockdown on GES-1 cell migration. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 14

The TFF3-JAK2/STAT3-CDX2 pathway as a feature of intestinal metaplasia. (A) Representative IHC images of TFF3, JAK2, STAT3, and CDX2 in normal gastric tissue and intestinal metaplasia tissue. Scale bar: 50 μm; (B) Quantitative analysis of IHC results for TFF3, JAK2, STAT3, and CDX2 across 15 samples of normal gastric tissue and intestinal metaplasia tissue. ****P < 0.0001.

Fig. 15

Correlation analysis between TFF3, JAK2, STAT3, and CDX2 expressions.