The regulatory effect of miR-101 and miR-125a-5p on autophagy in lipopolysaccharide derived THP-1 macrophages
10.3760/cma.j.issn.2095-4352.2016.04.009
- VernacularTitle:微小RNA-101和微小RNA-125a-5p在脂多糖诱导人THP-1巨噬细胞自噬中的调控作用
- Author:
Jieling XU
;
Zhenhui ZHANG
;
Huilin JIANG
;
Peiyi LIN
;
Xiaohui CHEN
- Publication Type:Journal Article
- Keywords:
micro-RNA;
Autophagy;
Macrophage;
Lipopolysaccharide
- From:
Chinese Critical Care Medicine
2016;28(4):334-338
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the role of micro-RNAs (miR-101 and miR-125a-5p) in autophagy of lipopolysaccharide (LPS) derived human THP-1 macrophages.Methods Human monocytic leukemia cell line THP-1 was cultured in vitro,and it was differentiated into macrophages after being induced with phorbol (50 μg/L) for 48 hours.THP-1 macrophages were stimulated with LPS in 0,250,500,1 000 μg/L respectively for 12 hours,miR-mimic was transfected into THP-1 macrophages as induced by Lipofectamine RNAiMAX,and the transfection efficiency of miRNA was determined with fluorescence microscopy.Enzyme linked immunosorbent assay (ELISA) was used to determine the levels of tumor necrosis factor-α (TNF-α) and monocyte chemotaxis protein-1 (MCP-1) in the supernatants of culture.Western Blot was used to detect the protein expressions of autophagy proteins ATG4D,Beclin1,and LC3 Ⅱ.The expression levels of miR-101 and miR-125a-5p were determined by quantitative reverse transcription-quantitative polymerase chain reaction (RT-qPCR).Results ① The releasing levels of TNF-α and MCP-1 induced by LPS with 250,500,1 000 μg/L were significantly increased as compared the cells without LPS stimulation [TNF-α (ng/L):1 336.1 ± 18.5,1 340.6±24.8,1 364.5± 14.9 vs.47.6±4.4;MCP-1 (ng/L):996.3 ±51.3,934.6±84.3,974.2±69.5 vs.21.3±6.5,all P ＜ 0.01],but no significant differences were found among the three LPS stimulation groups.The protein expressions of ATG4D,Beclin1 and LC3 1Ⅱ were up-regulated in the presence of different LPS concentrations (0,250,500,1 000 μg/L) for 12 hours in THP-1 macrophages (when compared with the cells without LPS stimulation,t value of ATG4D was 8.103,38.410,52.020,P value was 0.015,0.001,＜ 0.001;t value of Beclin1 was 3.026,5.328,3.482,P value was 0.047,0.034,0.037;t value of LC3 Ⅱ was 3.836,6.200,4.665,P value was 0.018,0.003,0.010),and the optimal concentration was 500 tg/L LPS.When THP-1 macrophages were stimulated with 500 μg/L LPS for 12 hours,the expression levels of miR-101 and miR-125a-5p were down-regulated significantly as compared with the cells without LPS stimulation [miR-101 (2-△ △Ct):0.68 ± 0.08 vs.1.95 ±0.26,t =8.047,P =0.001;miR-125a-5p (2-△ △Ct):0.23 ± 0.06 vs.1.69± 0.42,t =5.975,P =0.004].② The higher transfection efficiency was showed under fluorescence microscope.Westem Blot results showed the protein expressions of ATG4D,Beclin1 and LC3 Ⅱ were down-regulated as induced by an over-expression of miR-101 or miR-125a-5p in THP-1 macrophages,and more obviously down regulated by co-transfected with miR-101 and miR-125a-5p (compared with negative control group,t value was 14.550,5.855,14.180,P value was 0.005,0.014,＜ 0.001).Conclusion miR-101 and miR-125a-5p can inhibit the autophagy in LPS challenged THP-1 macrophages,and the potential mechanism might be related to target regulation of ATG4D.
