摘要
目的探讨脂多糖（LPS）诱导人THP-1巨噬细胞自噬过程中微小RNA-101和微小RNA-125a-5p（miR-101、miR-125a-5p）的调控作用。方法体外培养人白血病单核细胞THP-1，用佛波醇（PMA，50μg/L）诱导THP-1细胞48h分化成巨噬细胞，以0、250、500、1000μg/L梯度LPS刺激细胞12h，以转染试剂脂质体Lipofectamine RNAiMAX介导miRNA模拟物（miR—mimic）转染细胞，荧光显微镜下观察miRNA的转染效率。用酶联免疫吸附试验（ELISA）检测培养上清液中肿瘤坏死因子-α（TNF-α）、单核细胞趋化蛋白-1（MCP-1）的释放量；采用蛋白质免疫印迹试验（Western Blot）检测细胞内自噬相关蛋白ATG4D、Beclin1、LC3Ⅱ的蛋白表达；采用定量反转录-聚合酶链反应（RT—qPCR）检测miR-101和miR-125a-5p的表达。结果①250、500、1000μg/L LPS刺激THP-1巨噬细胞12h后，TNF-α和MCP-1释放量较未用LPS刺激细胞显著升高[TNF—α（ng／L）：1336．1±18．5、1340．6±24．8、1364．5±14．9比47．6±4．4，MCP-1（ng／L）：996．3±51．3、934．6±84．3、974．2±69．5比21．3±6．5，均P〈0．01]，但3个剂量组间无统计学差异。250、500、1000μg／L LPS刺激细胞12h后，细胞内ATG4D、Beclin1、LC3Ⅱ蛋白表达均较未用LPS刺激细胞明显上调（ATG4D的t值分别为8．103、38．410、52．020，P值分别为0．015、0．001、〈0．001；Beclin1的f值分别为3．026、5．328、3．482，P值分别为0．047、0．034、0．037；LC3Ⅱ的t值分别为3．836、6．200、4．665，P值分别为0．018、0．003、0．010），以500μg/L LPS的效果最为明显。用500峨几LPS刺激细胞12h后，细胞内miR-101、miR-125a-5p的表达均较未用LPS刺激细胞显著下调[miR-101（2^-△△Ct）：0．68±0．08比1．95±0．26，t=8．047，P=0．001；miR-125a-5p（2^-△△Ct）：0．23±0．06比1．69±0．42，t=5．975，P=0．004]。②荧光显微镜下显示miRNA转染效率较高。Western Blot结果显示，在细胞内分别过表达miR-101或miR-125a-5p后均可引起ATG4D、Beclin1、LC3Ⅱ蛋白表达一定程度的下调；但同时过表达miR-101和miR-125a-5p两个miRNAs时，ATG4D、Beclin1、LC3Ⅱ的蛋白表达下调更为显著（与阴性对照组比较t值分别为14．550、5．855、14．180，P值分别为0．005、0．014、〈0．001）结论miR-101、miR-125a-5p能抑制LPS诱导THP-1巨噬细胞自噬，其机制可能是通过靶向调节ATG4D完成。
Objeαive To investigate the role of micro-RNAs （miR-101 and miR-125a-5p） in autophagy of lipopolysaccharide （LPS） derived human THP-1 maerophages. Methods Human monoeytic leukemia cell line THP-1 was cultured in vitro, and it was differentiated into maerophages 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 respeαively for 12 hours, miR-mimic was transfeαed into THP-1 macrophages as induced by Lipofeαamine RNAiMAX, and the transfeαion efficiency of miRNA was determined with fluorescence microscopy. Enzyme linked immunosorbent assay （ELISA） was used to determine the levels of tumor necrosis faαor- α （TNF- α ） and monocyte ehemotaxis protein-1 （MCP-1） in the supernatants of culture. Western Blot was used to deteα the protein expressions of autophagy proteins ATG4D, Beclinl, and LC3 Ⅱ. The expression levels of miR-101 and miR-125a-5p were determined by quantitative reverse transcription-quantitative polymerase chain reaαion （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, Beclinl and LC3Ⅱ 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 Beclinl 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 μg/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. Western Blot results showed the protein expressions of ATG4D, Beclinl and LC3 Ⅱ were down-regulated as induced by an over-expression of miR-101 or miR-125a-5p in THP-1 maerophages, and more obviously down regulated by eo-transfeαed 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.
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2016年第4期334-338,共5页
Chinese Critical Care Medicine
基金
广东省自然科学基金（S2012010008971,S2013010014887）
广东省产业技术研究与开发资金计划项目（20128031800233）
广东省广州市医学重点学科建设项目（2013-16）
关键词
微小RNA
自噬
巨噬细胞
脂多糖
micro-RNA
Autophagy
Macrophage
Lipopolysaccharide
