MiR-3613-5p targets AQP4 to promote the progression of chronic atrophic gastritis to gastric cancer

Abstract

Introduction: Gastric cancer (GC) exhibits high invasiveness, delayed diagnosis, and poor prognosis. Chronic atrophic gastritis (CAG), an initial stage within the Correa cascade, induces gastric mucosal inflammation and atrophy, promoting genetic and epigenetic alterations. MicroRNAs (miRNAs) dysregulation has been implicated in gastric tumorigenesis, yet their specific roles in CAG progression to GC remain unclear. Methods: Using clinical data from the GEO database, we identified miRNAs differentially expressed in gastric mucosa and serum samples from GC patients. Murine CAG models were established through administration of N-methyl-N-nitrosourea (MNU) and high-salt diet (HSD). In vitro functional assays evaluated proliferation and migration after miRNA modulation in gastric cancer cell lines. MiRNA target validation involved luciferase reporter assays. Results: MiR-3613-5p expression was significantly elevated in gastric mucosal and serum samples of GC patients, mucosal tissues of CAG patients, tumor tissues, and human gastric cancer cell lines. Murine models demonstrated increased miR-3613-5p expression in gastric mucosa following MNU and HSD-induced CAG. Functionally, miR-3613-5p overexpression promoted gastric cancer cell proliferation and migration in vitro, whereas silencing miR-3613-5p alleviated pathological gastric mucosal alterations (atrophy, hyperplasia, inflammatory infiltration) in vivo. Mechanistically, miR-3613-5p inhibited Aquaporin 4 (AQP4) expression by directly targeting its 3'UTR. Discussion: Our findings provide the first evidence that miR-3613-5p facilitates CAG progression toward GC via negative regulation of AQP4. These results highlight miR-3613-5p as a promising biomarker and therapeutic target, suggesting antagomiR-3613-5p as a potential novel strategy to prevent gastric carcinogenesis.

Keywords: Aquaporin 4; chronic atrophic gastritis; gastric cancer; intestinal metaplasia; miR-3613-5p.

FIGURE 1

miR3613-5p is significantly overexpressed in human CAG and gastric cancer tissues. (A) Venn diagram of significantly upregulated miRNAs in the GSE186595 and GSE211692 datasets. A total of 72 differentially expressed genes (DEGs) for microRNAs were significantly upregulated in the gastric mucosal of gastric cancer patients compared to healthy controls in the GSE186595 dataset (green), while 704 DEGs were significantly upregulated in the serum of early gastric cancer patients compared to healthy controls in the GSE211692 dataset (purple). There are 25 microRNAs that are differentially expressed in both datasets (yellow), all of which are displayed below, including miR-3613. (B, C) Box plot of miR-3613-5p expression level in GSE186595 (B) and GSE211692 (C) dataset. (D) Relative expression levels of miR-3613-5p in gastric mucosal from CAG patients and healthy individuals (n = 8). (E) Relative expression levels of miR-3613-5p in gastric cancer and adjacent non-tumor tissues (n = 6). Data are presented as mean ± SEM. Statistical analysis is performed using a Student’s t-test and statistical significance is shown as *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 2

miR3613-5p is significantly overexpressed in gastric cancer cell lines and the gastric mucosa of CAG mice. (A) Relative expression levels of miR-3613-5p in human gastric cancer cell lines (BGC-823, MKN-45, SGC-7901) and the normal gastric mucosal epithelial cell line GES-1. (B) Schematic diagram of the modeling method for CAG mice. MNU was added to the drinking water for the mice to drink freely, and a 1mL saturated NaCl solution was administered by gavage every 3 days, continuing for 12 weeks. (C) Representative H&E staining images of gastric mucosal slices from control and MNU + HSD mice after 12 weeks of induction. (D-E) Levels of IL-6 and TNF-α in the serum of control and MNU + HSD mice detected by ELISA (n = 5). (F) Relative expression levels of miR-3613-5p in the gastric mucosa of control and MNU + HSD mice (n = 5). Data are presented as mean ± SEM. Statistical analysis is performed using a Student’s t-test and statistical significance is shown as *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 3

miR-3613-5p promotes the proliferation and migration of gastric cancer cells. (A–B) Relative expression levels of miR-3613-5p after transfection of AgomiR-3613-5p and AntagomiR-3613-5p in SGC-7901 cell line. (C, E) Representative images of Ki-67 immunofluorescence staining after transfection of AgomiR-3613-5p and AntagomiR-3613-5p in SGC-7901 cell line. (D, F) Statistical analysis of the percentage of Ki-67 positive cells after transfection of AgomiR-3613-5p and AntagomiR-3613-5p. (G, I). Representative images from the cell wound healing assay after transfection of AgomiR-3613-5p and AntagomiR-3613-5p. (H, J) Statistical analysis of the wound healing assay. The migration index was calculated to evaluate the cell migration capacity. Migration index = (A0h - A24h)/A0h. Scratch area (Area, A) was measured by ImageJ software (version 1.52a; National Institutes of Health). Every experiment was repeated 3 times. Data are presented as mean ± SEM. Statistical analysis is performed using a Student’s t-test and statistical significance is shown as *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 4

Overexpression of miR-3613-5p promotes the progression from CAG to gastric cancer in mice. (A, E) Relative expression levels of miR-3613-5p in gastric mucosa of CAG mice after tail vein injection of AgomiR-3613-5p or AntagomiR-3613-5p. (B, F) Representative images of H&E staining of mouse gastric mucosal slices. (C, G) Levels of IL-6 in mouse serum detected by ELISA (n = 5). (D, H) Levels of TNF-α in mouse serum detected by ELISA (n = 5). Data are presented as mean ± SEM. Statistical analysis is performed using a Student’s t-test and statistical significance is shown as *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 5

miR-3613-5p inhibits the expression of the AQP4 gene by binding to its 3′UTR. (A) The Venn diagram illustrates the overlap between the target genes predicted by TargetScan 8.0 (red) and the downregulated genes in the gastric mucosa of gastric cancer patients from the GSE130823 dataset (blue). The 39 presumed target genes in the intersection are listed below, including AQP4. (B) Relative mRNA expression levels of PTGER3, HSPB7, AQP4, ERBB4, and PAQR5 in SGC-7901 cell line after transfection with AgomiR-NC and AgomiR-3613-5p. (C) Pearson correlation coefficient between AQP4 and miR-3613-5p. Data from GSE224056. (D) Predicted binding sites of miR-3613-5p in the 3′ UTR of the AQP4 gene according to TargetScan 8.0. (E) Relative luciferase activity after co-transfection of SGC-7901 cells with pGL3, pGL3-AQP4-WT, pGL3-AQP4-MUT, as well as AgomiR-NC and AgomiR-3613-5p. Data are presented as mean ± SEM. Statistical analysis is performed using a Student’s t-test and statistical significance is shown as *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 6

miR-3613-5p promotes the progression from CAG to gastric cancer by inhibiting the expression of AQP4. (A) Protein levels of AQP4 after transfection of AgomiR-NC or AgomiR-3613-5p in SGC-7901 cells. (B) Quantification of protein expression in (A). (C) Protein levels of AQP4 in the gastric mucosa 1 month after tail vein injection of AgomiR-NC or AgomiR-3613-5p in MNU + HSD mice. (D) Quantification of protein expression in (C) Data are presented as mean ± SEM. Statistical analysis is performed using a Student’s t-test and statistical significance is shown as *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 7

Model on how miR-3613-5p promotes the progression from CAG to gastric cancer. (A) miR-3613-5p inhibits the expression of the AQP4 gene by binding to its 3′ UTR, thereby promoting the progression from CAG to gastric cancer.