Inhibitory effect of miR-487a on M2-type polarization of gastric cancer tumor-associated macrophages by targeting TIA1
10.13481/j.1671-587X.20240317
- VernacularTitle:miR-487a通过靶向调控TIA1对胃癌肿瘤相关巨噬细胞M2型极化的抑制作用
- Author:
Yan QU
1
;
Lin DAI
;
Biao WANG
;
Duji RUAN
;
Yuchang ZHONG
;
Xuefeng YANG
Author Information
1. 遵义医科大学第二附属医院胃肠外科,贵州 遵义 563006
- Keywords:
Gastric neoplasm;
MicroRNA-487a;
T-lymphocyte-restricted intracellular antigen-1;
Tumor-associated macrophage;
M2 polarization
- From:
Journal of Jilin University(Medicine Edition)
2024;50(3):728-738
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To discuss the inhibitory effect of microRNA-487a(miR-487a)on the M2 polarization of tumor-associated macrophages(TAMs)in gastric cancer,and to clarify its effect on the proliferation,invasion,and migration of the gastric cancer AGS cells.Methods:The TAMs from gastric cancer tissue and adjacent normal tissue macrophages(NTMs)from adjacent tissue of the primary gastric cancer patients were isolated and cultured.The human monocyte THP-1 cells were induced in vitro to differentiate into TAMs,and the differentiated M0,M1,and M2 macrophages were cultured for 24 h by conditioned medium(CM)to obtain the TAMs,M1-TAMs,and M2-TAMs respectively.The TAMs were transfected and then divided into blank group,inhibitor-NC group,miR-487a inhibitor group,miR-487a inhibitor+si-NC group,and miR-487a inhibitor+si-TIA1 group.The transfection efficiencies of the cells in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods.The M2-TAMs were co-cultured with the AGS cells,and divided into AGS group,AGS+inhibitor-NC group,AGS+miR-487a inhibitor group,AGS+miR-487a inhibitor+si-NC group,and AGS+miR-487a inhibitor+si-TIA1 group.RT-qPCR method was used to detect the expression levels of miR-487a and lymphocyte intracytoplasmic antigen-1(TIA1)mRNA in TAMs from gastric cancer tissue and NTMs from adjacent normal tissue in various groups;Western blotting method was used to detect the expression level of TIA1 protein in TAMs from gastric cancer tissue and NTMs from adjacent normal tissue and TAMs in various groups;flow cytometry was used to detect the levels of CD206 and CD163 in TAMs in various groups;enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-10(IL-10),transforming growth factor-beta(TGF-β),vascular endothelial growth factor A(VEGF-A),and arginase-1(Arg-1)in culture supernatant of the TAMs cells;CCK-8 assay was used to detect the proliferative activity of the AGS cells in various groups;wound healing assay was used to detect the migration rates of the AGS cells in various groups;Transwell assay was used to detect the number of invasion AGS cells in various groups.Results:The RT-qPCR results shoued that compared with NTMs from adjacent tissue,the expression level of miR-487a in the TAMs from gastric cancer tissue was significantly increased(P<0.01)and the expression level of TIA1 mRNA was significantly decreased(P<0.01).Compared with TAMs,the expression level of miR-487a in M1-TAMs was significantly decreased(P<0.01),and the expression level of TIA1 mRNA was increased(P<0.01);the expression level of miR-487a in M2-TAMs was significantly increased(P<0.01),and the expression level of TIA1 mRNA was decreased(P<0.01).After transfection,compared with blank group and inhibitor-NC group,the expression level of miR-487a in the cells in miR-487a inhibitor group was significantly decreased(P<0.01),indicating successful transfection.The Western blotting results showed that compared with NTMs from adjacent normal tissue,the expression level of TIA1 protein in TAMs from gastric cancer tissue was decreased(P<0.01);compared with TAMs,the expression level of T1A1 protein in M1-TAMs was significantly increased(P<0.01),and the expression of TIA1 protein in M2-TAMs was significantly decreased(P<0.01);after co-transfection,compared with inhibitor-NC group,the expression level of TIA1 protein in the cells in miR-487a inhibitor group was significantly increased(P<0.01);compared with miR-487a inhibitor+si-NC group,the expression level of TIA1 protein in the cells in miR-487a inhibitor+si-TIA1 group was significantly decreased(P<0.01).The flow cytometry results showed that compared with blank group and inhibitor-NC group,the levels of CD206 and CD163 in the cells in miR-487a inhibitor group were significantly decreased(P<0.01);after co-transfection,compared with inhibitor-NC group,the levels of CD206 and CD163 in the cells in miR-487a inhibitor group were significantly decreased(P<0.01);compared with miR-487a inhibitor+si-NC group,the levels of CD206 and CD163 in the cells in miR-487a inhibitor+si-TIA1 group were significantly increased(P<0.01).The ELISA results showed that compared with blank group and inhibitor-NC group,the levels of IL-10,TGF-β,VEGF-A,and Arg-1 in culture supernatant of the TAMs in miR-487a inhibitor group were significantly decreased(P<0.01);after co-transfection,compared with inhibitor-NC group,the levels of IL-10,TGF-β,VEGF-A,and Arg-1 in culture supernatant of the TAMs in miR-487a inhibitor group were significantly decreased(P<0.01);compared with miR-487a inhibitor+si-NC group,the levels of IL-10,TGF-β,VEGF-A,and Arg-1 in culture supernatant of the TAMs in miR-487a inhibitor+si-TIA1 group were significantly increased(P<0.01).The CCK-8 assay results showed that compared with AGS group,the proliferation activity of the cells in AGS+inhibitor-NC group was significantly increased(P<0.01);compared with AGS+inhibitor-NC group,the proliferation activity of the cells in AGS+miR-487a inhibitor group was significantly decreased(P<0.01);compared with AGS+miR-487a inhibitor+si-NC group,the proliferation activity of the cells in AGS+miR-487a inhibitor+si-TIA1 group was significantly increased(P<0.01).The wound healing assay results showed that compared with AGS group,the migration rate of the cells in AGS+inhibitor-NC group was significantly(P<0.05);compared with AGS+inhibitor-NC group,the migration rate of the cells in AGS+miR-487a inhibitor group was significantly decreased(P<0.01);compared with AGS+miR-487a inhibitor+si-NC group,the migration rate of the cells in AGS+miR-487a inhibitor+si-TIA1 group was significantly increased(P<0.05).The Transwell assay results showed that compared with AGS group,the number of invasion AGS cells in AGS+inhibitor-NC group was significantly increased(P<0.01);compared with AGS+inhibitor-NC group,the number of invasion AGS cells in AGS+miR-487a inhibitor group was significantly decreased(P<0.01);compared with AGS+miR-487a inhibitor+si-NC group,the number of invasion AGS cells in AGS+miR-487a inhibitor+si-TIA1 group was significantly increased(P<0.01).Conclusion:Silencing the miR-487a expression can inhibit the M2 polarization of the gastric cancer-associated macrophages by targeted upregulation of TIA1,and suppress the proliferation,migration,and invasion of the gastric cancer cells.
