ABSTRACT
OBJECTIVE: To investigate the association between Helicobacter pylori infection and GDF6 expression in gastric cancer patients, and to determine its influence on prognosis and resistance to capecitabine. METHODS: Tumor and adjacent non-tumor tissues were collected from 148 gastric cancer patients who underwent surgery in our department from October 2019 to June 2022. Of these patients, 78 tested positive for Helicobacter pylori and 70 tested negative. Hematoxylin-eosin (HE) and immunofluorescence staining were utilized to quantify GDF6 expression in cancerous and adjacent tissues. Patient prognosis was monitored via follow-up. Western blotting analyzed GDF6 expression in common gastric cancer cell lines. HGC27 cells exhibiting high GDF6 expression and BGC823 cells with low expression were used to create GDF6-silenced and overexpressed cell lines. The impact of GDF6 on the proliferation, migration, invasion, and cloning abilities of gastric cancer cells was evaluated using the CCK-8 assay, scratch test, Transwell assay, and plate colony formation assay. Fluorescent quantitative PCR and Western blotting assessed the effects of GDF6 levels on epithelial-mesenchymal transition (EMT) and tumor cell stemness. RESULTS: GDF6 expression in gastric cancer tissues was significantly correlated with cancer grading and staging (P<0.05). Helicobacter pylori-positive tissues exhibited significantly higher GDF6 expression levels than negative samples (P<0.05). Kaplan-Meier survival analysis indicated that high GDF6 expression was associated with poor survival prognosis. Overexpressed GDF6 enhanced the proliferation, migration, and invasion abilities of gastric cancer cells, while silencing GDF6 yielded opposite results. Increased GDF6 expression upregulated TGF-ß expression and the phosphorylation levels of SMAD3, leading to an elevation in mesenchymal cell markers N-cadherin, vimentin, and a reduction in epithelial cell markers cytokeratins, E-cadherin. Moreover, high GDF6 levels contributed to increased resistance to capecitabine and enhanced the expression of tumor stem cell markers Nanog, Sox-2, Oct-4, CD44, amplifying tumor cell stemness. CONCLUSION: Helicobacter pylori infection is associated with increased GDF6 expression in gastric cancer tissue, correlating with poor survival prognosis. Elevated GDF6 expression promotes the proliferation, migration, and invasion abilities of gastric cancer cells, facilitates EMT via the TGF-ß/SMAD3 pathway, and intensifies cell stemness and capecitabine resistance. Consequently, GDF6 presents itself as a potential new target for gastric cancer treatment. DATA AVAILABILITY STATEMENT: The data that support the findings of this study are available from the corresponding author upon reasonable request.
ABSTRACT
Gastric cancer (GC) is the second deadliest disease in Asia, so it is crucial to find its promising therapeutic targets. The expression profile data of miR383-5p in the Cancer Genome Atlas (TCGA) were analyzed. The expression levels of miR383-5p in the collected clinical tissue samples and peripheral blood samples were examined by qPCR, and the relationship between its expression and the clinical data of patients was evaluated. MiR383-5p was overexpressed in the AGS cells, and cell biology assays, such as Transwell, were performed to detect the cell proliferation, migration, invasion and other cell biology abilities of miR383-5p. Target prediction and dual luciferase reporter gene assay were performed to find and validate the target genes of miR383-5p. The expression and activity of MMP and related proteins after overexpression of miR383-5p and NCKAP1 were detected by WB and gelatin zymography assay. The expression of miR383-5p was down-regulated in GC tissues, and its low expression was associated with lymph node metastasis. Restoration of miR383-5p expression in GC cells can inhibit the invasion and migration abilities of GC cells. MiR383-5p negatively regulated NCKAP1 through direct interaction with the 3'UTR sequence of NCKAP1. The overexpression of NCKAP1 can improve the migration and invasion abilities of GC cells, whereas overexpression of miR383-5p can inhibit growth of the aforementioned abilities of GC cells induced by NCKAP1 overexpression. The overexpression of NCKAP1 can increase the expression level and activity of MMP2, while the overexpression of miR383-5p can inhibit the increase of MMP2 expression level and activity in GC cells induced by NCKAP1 overexpression. NCKAP1 is a target gene of miR383-5p, and miR383-5p could be a valuable therapeutic target for stomach adenocarcinoma.
