S100 calcium binding protein A16 promotes fat synthesis through endoplasmic reticulum stress in HepG2 cells
S100 calcium binding protein A16 promotes fat synthesis through endoplasmic reticulum stress in HepG2 cells
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DOI：
10.13294/j.aps.2019.0027
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发表时间：
2019-04-01
期刊：
影响因子：
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通讯作者：
Liu Yun
中科院分区：
文献类型：
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作者：
Kan Jing-Bao;Shen Ge-Qian;Liu Yun
本研究旨在探讨S100钙结合蛋白A16 （S100A16）在肝细胞脂质代谢中的作用及其可能的生物学机制。用脂肪酸培养人肝癌细胞系HepG2细胞，建立脂肪酸培养模型。对照模型不加脂肪酸培养。每个模型分为三组，分别转染S100a16过表达质粒、shRNA质粒和载体质粒。用试剂盒测定细胞内甘油三酯（TG）浓度，油红O染色观察脂滴。利用免疫沉淀法和质谱法寻找与S100A16相互作用的感兴趣蛋白，并通过免疫沉淀法验证相互作用。通过Western blot和qRT-PCR进一步研究其作用机制。结果表明，脂肪酸培养模型细胞内脂滴和TG浓度显著高于对照模型。S100a16过表达组细胞内脂肪积累量显著高于载体质粒转染组。热休克蛋白A5 （HSPA5）与S100A16存在相互作用。S100A16的过表达上调HSPA5、肌醇要求酶1 α (IRE1 α)和pIRE α 1的蛋白表达水平，属于内质网应激HSPA5/IRE1 α - xbp1通路。同时，过表达S100A16上调脂肪合成相关基因Srebplc、Acc和Fas的mRNA表达水平。S100a16 shRNA质粒转染组上述蛋白和mRNA水平均低于载体质粒转染组。提示S100A16可能通过内质网应激HSPA5/IRE1 α - xbp1途径促进HepG2细胞脂质合成。
The aim of this study was to investigate the role of S100 calcium binding protein A16 (S100A16) in lipid metabolism in hepatocytes and its possible biological mechanism. HepG2 cells (human hepatoma cell line) were cultured with fatty acid to establish fatty acid culture model. The control model was cultured without fatty acid. Each model was divided into three groups and transfected with S100a16 over-expression, shRNA and vector plasmids, respectively. The concentration of triglyceride (TG) in the cells was measured by kit, and the lipid droplets was observed by oil red O staining. Immunoprecipitation and mass spectrometry were used to find the interesting proteins interacting with S100A16, and the interaction was verified by immunoprecipitation. The further mechanism was studied by Western blot and qRT-PCR. The results showed that the intracellular lipid droplet and TG concentrations in the fatty acid culture model were significantly higher than those in the control model. The accumulation of intracellular fat in the S100a16 over-expression group was significantly higher than that in the vector plasmid transfection group. There was an interaction between heat shock protein A5 (HSPA5) and S100A16. Over-expression of S100A16 up-regulated protein expression levels of HSPA5, inositol-requiring enzyme 1 alpha (IRE1 alpha) and pIRE alpha 1, which belong to endoplasmic reticulum stress HSPA5/IRE1 alpha-XBP1 pathway. Meanwhile, over-expression of S100A16 up-regulated the mRNA expression levels of adipose synthesis-related gene Srebplc, Acc and Fas. In the S100a16 shRNA plasmid transfection group, the above-mentioned protein and mRNA levels were lower than those of vector plasmid transfection group. These results suggest that S100A16 may promote lipid synthesis in HepG2 cells through endoplasmic reticulum stress HSPA5/IRE1 alpha-XBP1 pathway.