S100钙结合蛋白A16通过内质网应激促进HepG2细胞脂肪合成
阚敬保¹, 沈歌前¹, 杨洁¹, 童佩¹, 张日华¹, 梁秀斌², 苏东明², 李东³, 刘云^1,*
1南京医科大学第一附属医院老年医学科，南京 210029；²南京医科大学代谢病研究中心，南京 210029；³南京中医药大学附属江苏省中医院整形外科，南京 210029
摘要
本研究旨在探讨S100钙结合蛋白A16 (S100 calcium binding protein A16, S100A16)在肝细胞脂质代谢中的作用及可能的生物学机制。用脂肪酸培养HepG2细胞(人肝癌细胞系)以建立脂肪酸培养模型，对照模型不加脂肪酸培养，每一模型分成3组细胞，分别用S100a16过表达质粒、shRNA质粒、Vector质粒转染。用试剂盒检测细胞甘油三酯浓度，用油红O染色观察脂滴聚集情况，用免疫沉淀和质谱分析寻找和S100A16相互作用的兴趣蛋白，并用免疫共沉淀验证，用Western blot和qRT-PCR进行相关机制研究。结果显示，和对照模型相比，脂肪酸培养模型细胞内脂肪和甘油三酯浓度显著增加。S100a16过表达组细胞内脂肪积累显著高于Vector质粒转染组。热休克蛋白A5 (heat shock protein A5, HSPA5)与S100A16之间存在相互作用。S100a16过表达可上调内质网应激的HSPA5/肌醇依赖酶1α-X结合蛋白1 (inositol-requiring enzyme 1α-X binding protein 1, IRE1α-XBP1)通路相关蛋白(HSPA5、IRE1α和pIREα1)表达水平，并上调脂肪合成相关基因Srebp1c、Acc和Fas mRNA表达水平，而转染S100A16 shRNA质粒可使上述蛋白和mRNA水平低于Vector质粒转染组。以上结果提示，S100A16可能通过内质网应激HSPA5/IRE1α-XBP1通路促进HepG2细胞脂质合成。
关键词： S100A16; 脂代谢; HSPA5; 内质网应激
分类号：R33

S100 calcium binding protein A16 promotes fat synthesis through endoplasmic reticulum stress in HepG2 cells
KAN Jing-Bao¹, SHEN Ge-Qian¹, YANG Jie¹, TONG Pei¹, ZHANG Ri-Hua¹, LIANG Xiu-Bin², SU Dong-Ming², LI Dong³, LIU Yun^1,*
1Department of Geratology, The First? Affiliated? Hospital? of? Nanjing? Medical? University, Nanjing? 210029, China；²The Center of Metabolic Disease Research, Nanjing Medical University, Nanjing 210029, China；³Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China
Abstract
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 S100a16over-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 1α (IRE1α) and pIREα1, which belong to endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway. Meanwhile, over-expression of S100A16 up-regulated the mRNA expression levels of adipose synthesis-related gene Srebp1c, 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/IRE1α-XBP1 pathway.
Key words: S100A16; lipid metabolism; HSPA5; endoplasmic reticulum stress

收稿日期：2018-10-15　　录用日期：2018-11-14
通讯作者：刘云　　E-mail: liuyun@njmu.edu.cn
DOI: 10.13294/j.aps.2019.0027
引用本文：
阚敬保, 沈歌前, 杨洁, 童佩, 张日华, 梁秀斌, 苏东明, 李东, 刘云. S100钙结合蛋白A16通过内质网应激促进HepG2细胞脂肪合成[J]. 生理学报 2019; 71 (2): 279-286.
KAN Jing-Bao, SHEN Ge-Qian, YANG Jie, TONG Pei, ZHANG Ri-Hua, LIANG Xiu-Bin, SU Dong-Ming, LI Dong, LIU Yun. S100 calcium binding protein A16 promotes fat synthesis through endoplasmic reticulum stress in HepG2 cells. Acta Physiol Sin 2019; 71 (2): 279-286 (in Chinese with English abstract).



PDF全文下载地址:

点我下载PDF