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Effects of miR-133a-5p on the proliferation and adhesion of gastric cardia adenocarcinoma cells and the polarization of M1 macrophages
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Abstract:
Objective: To investigate the effect of miR-133a-5p regulating serum exosomes derived fibronectin1 (FN1) on the proliferation, adhesion, and M1 macrophage polarization of gastric cardia adenocarcinoma (GCA) cells. Methods: The differentially expressed genes in GCA were predicted using the GEO database, and their functional enrichment analysis was performed. qPCR was used to detect the expression of FN1 in GCA tissue, serum and serum-derived exosomes of GCA patients, and GCA cells. The FN1 overexpression vector and its control plasmid were respectively transfected into the serum-derived exosomes of GCA patients. The miR-133a-5p mimics and its control mimics were transfected into HGC-27 cells. The transfected HGC-27 cells were co-cultured with transfected exosomes, and then the cells were co-cultured with THP-1 cells. CCK-8 and cell adhesion experiments were used to detect the proliferation and adhesion of HGC-27 cells in each group. WB method and ELISA were used to detect the levels of CD86 and iNOS in cells and the effects of transfection on the secretion of IL-6 and IL-1β from macrophages. Dual-luciferase reporter experiment was adopted to verify the interaction between FN1 mRNA and miR-133a-5p. Results: Compared with healthy controls, the expression levels of FN1 in GCA tissues, serum and serum-derived exosomes from GCA patients, and GCA cells were significantly up-regulated (all P<0.05). High expression of FN1 in serum exosomes was associated with the TNM stage (P=0.032 9) and lymph node metastasis (P=0.012 7) in GCA patients. FN1-riched exosomes could be internalized by GCA cells, and co-culture with FN1-riched exosomes could improve the proliferation and adhesion ability of GCA cells and inhibit the polarization of M1-type macrophages by blocking the expression of IL-6, IL-1β, CD86, and iNOS (P<0.05 or P<0.01). miR-133a-3p was lowly expressed in GCA tissues and cells and could negatively regulate the expression of FN1. Overexpression of miR-133a-5p could promote the expression of IL-6, IL-1β,CD86, and iNOS in M1 macrophages by reducing the proliferation and adhesion of GCA cells, and partially reversing the promotion effect of FN1 on the malignant behaviors of GCA cells (P<0.05 or P<0.01). Conclusion: miR-133a-5p inhibits the proliferation and adhesion of GCA cells via suppressing the secretion of FN1 by serum exosomes and promotes the polarization of M1 macrophages.
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