Abstract
BackgroundCancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor microenvironment, have a pivotal role in tumor progression. Dysregulation of microRNAs (miRNAs) in CAFs contributes to the tumor-promoting ability of CAFs. However, the mechanism underlying the involvement of miRNAs in CAFs of gastric cancer (GC) is not fully understood. This study aimed to explore the effects of miRNA-214 in CAFs on GC migration and invasion.MethodsThe primary CAFs and corresponding normal fibroblasts (NFs) were isolated. Cell counting kit-8, EdU cell proliferation staining and Transwell assays were used to determine the role of miRNA-214 in GC progression. Real-time polymerase chain reaction, Western blot analysis, and dual-luciferase reporter assay were performed to verify the target genes of miRNA-214. Immunofluorescence and Western blot analysis were applied to detect the expression of epithelial–mesenchymal transition (EMT) markers. Immunohistochemistry and in situ hybridization were implemented to analyze the fibroblast growth factor 9 (FGF9) and miRNA-214 expression in human GC tissues, respectively. Finally, to assess its prognostic relevance, Kaplan–Meier survival analysis was conducted.ResultsMiRNA-214 was significantly downregulated in CAFs of GC compared with NFs. The upregulation of miRNA-214 in CAFs inhibited GC cell migration and invasion in vitro but failed to affect proliferation. Moreover, GC cells cultured with conditioned medium from CAFs transfected with miR-214 mimic showed increased expression of E-cadherin and decreased expression of Vimentin, N-cadherin and Snail, indicating the suppression of EMT of GC cells. Furthermore, FGF9 was proved to be a direct target gene of miR-214. The expression of FGF9 was higher in CAFs than that in tumor cells not only in primary tumor but also in lymph node metastatic sites (30.0% vs 11.9%, P < 0.01 and 32.1% vs 12.3%, P < 0.01, respectively). Abnormal expression of FGF9 in CAFs of lymph node metastatic sites was significantly associated with poor prognosis in patients with GC (P < 0.05).ConclusionsThis study showed that miR-214 inhibited the tumor-promoting effect of CAFs on GC through targeting FGF9 in CAFs and regulating the EMT process in GC cells, suggesting miRNA-214/FGF9 in CAFs as a potential target for therapeutic approaches in GC.
Highlights
Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor microenvironment, have a pivotal role in tumor progression
The expression of fibroblast biomarker in these primary cultured cells was examined to test the purity of normal fibroblasts (NFs) and CAFs
Western blot analysis and qRT-polymerase chain reaction (PCR) assay further confirmed that the protein and mRNA expression levels of Fibroblast activation protein (FAP) significantly increased in CAFs compared with NFs (Fig. 1b and c)
Summary
Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor microenvironment, have a pivotal role in tumor progression. Chemotherapy and targeted therapy (trastuzumab) benefit a part of patients, tumor invasion and metastasis remain major obstacles in treatment and prognosis. Stromal cells within the TME are genetically stable, making them a more attractive therapeutic target for cancer therapy. Cancer-associated fibroblasts (CAFs), one of the most important stromal cell types in TME, have been considered as co-conspirators of tumor progression [3, 4]. CAFs separated from GC tissues had an essential role in the migration and invasion of GC cells [8, 9]. The mechanisms underlying the tumor-promoting effect of CAFs on GC cells remain obscure
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