miR-133a-5p对胃贲门腺癌细胞增殖、黏附和M1型巨噬细胞极化的影响

Effects of miR-133a-5p on the proliferation and adhesion of gastric cardia adenocarcinoma cells and the polarization of M1 macrophages

目的:探究miR-133a-5p调控血清外泌体源性纤连蛋白1(fibronectin1,FN1)的表达对胃贲门腺癌(gastric cardia adenocarcinoma,GCA)细胞增殖、黏附和M1型巨噬细胞极化的影响。方法:借助GEO数据库分析GCA组织中的差异表达基因,进行功能富集分析。采用qPCR检测FN1在GCA组织、血清、血清外泌体和细胞中的表达。向GCA患者血清外泌体中转染FN1的过表达载体及其对照质粒,向HGC-27细胞中转染miR-133a-5p模拟物及其对照,将转染后的HGC-27细胞和外泌体共培养,再将此细胞与THP-1细胞共培养。采用CCK-8和细胞黏附实验分别检测各组HGC-27细胞的增殖和黏附情况,WB法、ELISA分别检测细胞中CD86、iNOS的水平以及对巨噬细胞分泌IL-6、IL-1β的影响。采用双荧光素酶报告基因实验验证FN1 mRNA和miR-133a-5p之间的相互作用关系。结果:与健康人对照组相比,GCA组织、血清、血清外泌体和细胞中的FN1表达水平显著上调(均P<0.05),FN1高表达的血清外泌体与GCA患者的TNM分期(P=0.0329)和淋巴结转移有关联(P=0.0127)。富含FN1的血清外泌体能够被GCA细胞内化,与过表达FN1的外泌体共培养能够提高HGC-27细胞的增殖和黏附能力,抑制THP-1细胞中IL-6、IL-1β、CD86和iNOS的表达,抑制M1型巨噬细胞极化(P<0.05或P<0.01)。miR-133a-5p在GCA组织和细胞中较对照组显著降低,可负调控FN1的表达,过表达miR-133a-5p能够通过降低GCA细胞增殖和黏附能力,促进IL-6、IL-1β、CD86和iNOS的表达,部分逆转FN1对GCA细胞恶性行为的促进作用(P<0.05或P<0.01)。结论:miR-133a-5p可通过抑制血清外泌体分泌FN1对GCA细胞的恶性行为起抑制作用,对M1型巨噬细胞极化起促进作用。

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-1βfrom 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=0.0329)and lymph node metastasis(P=0.0127)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.