蛋白质组分析油酸对HepG2细胞转录因子DNA结合活性的影响  

Proteome-wide Screening of Transcription Factor DNA Binding Activity in HepG2 Cells after Oleic Acid Treatment

作　　者：梁丽珠[1,2,3] 孙佳楠[3,4] 李恺[3,5] 刘明伟 丁琛[2,3] 秦钧[1,2,3] 

机构地区：[1]安徽医科大学研究生学院,合肥230032 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]国家蛋白质组学重点实验室北京蛋白质组学研究中心,北京102206 [4]沈阳药科大学药学院,沈阳110016 [5]河北联合大学生命科学学院,唐山063009

出　　处：《中国生物工程杂志》2015年第5期22-31,共10页China Biotechnology

基　　金：北京市自然科学基金资助项目(5132012)

摘　　要：目的:建立由于油酸大量摄入造成的HepG2细胞脂肪变性模型;发现HepG2细胞发生脂肪变性前、后转录因子DNA结合活性的差异。方法:将HepG2细胞在含2mmol/L油酸的培养基中培养24h,通过油红O染色鉴定细胞中的甘油三酯含量,并确定发生脂肪变性的程度。分别提取对照组和实验组的核蛋白,利用转录因子富集技术cat TFRE(a concatenated tandem array of the consensus transcription factor response element),同时运用深度覆盖的蛋白质组学手段,对HepG2细胞脂肪变性前、后的转录因子变化进行动态描绘和定量分析。最后,利用IPA(integrated pathway analysis)对DNA结合活性发生改变的转录因子进行功能分析。结果:对照组总共鉴定到170种转录因子,实验组总共鉴定到190种转录因子,两组总共鉴定到208种转录因子。HepG2细胞发生脂肪变性后,DNA结合活性发生变化的转录因子有101种(重复实验间差异小于2倍,不同处理间差异大于2倍),DNA结合活性加强的有67种,减弱的有34种。其中,调节糖代谢、脂代谢的关键转录因子MLX、MLXIPL的DNA结合活性减弱;NF-κB1的DNA结合活性增加,暗示了NF-κB介导的炎症反应的激活。IPA分析的结果表明,油酸激活了HepG2细胞内NRF2介导的氧化应激反应,促进了基因表达(gene expression)、细胞增殖(cell proliferation)、细胞分化(differentiation of cell)、细胞周期(cell cycle)及细胞存活(cell survival)的进程。结论:油酸诱导HepG2细胞脂肪变性会引起细胞内糖、脂代谢的紊乱,诱发炎症反应,并引起氧化损伤,但是在转录水平上,细胞通过加强脂类代谢基因的表达、减少糖类向脂肪酸的转化、促进细胞增殖及激活NRF2介导的氧化应激反应等,最终促进了HepG2细胞的存活。Nonalcoholic fatty liver disease （NAFLD）, one of the most prevalent chronic diseases worldwide, is associated with accumulation of fat in liver and is a manifestation of metabolic syndromes （including obesity, diabetes, and insulin resistance ）. HepG2 cells are selected to establish a model in vitro about hepatic steatosis and to identify the transcription factors （TFs） that change their binding affinity to DNA in response to hepatic steatosis. HepG2 cells are incubated with 2mmol/L oleic acid for 24 hours. Oil-Red-O staining is done to show triglycerides （TG） and assess the extent of hepatic steatosis. The nuclear extraction from each group is separated from the cytoplasmic extraction and the transcription factors in nuclear extraction are enriched by a concatenated tandem array of consensus transcription factor response element （cat TFRE ）. By combination of catTFRE and an in-depth analysis of proteomies expression profiling, the dynamic description and quantitative analysis of the transcription factors＇ DNA binding activity changes are shown. Then the functions of the altered transcription factors are analyzed by IPA（ integrated pathway analysis）. 170 TFs are identified in the control groups and 190 TFs are identified in the oleic acid treated groups. 208TFs are identified in all experiments. In the 208 TFs, DNA binding activity of 67 TFs are up-regulated obviously and 34 TFs are down- regulated obviously. The DNA binding activity of MLX and MLXIPL, which play important roles in glycometabolism, down-regulated obviously. The DNA binding activity of NF-KB1 is up-regulated obviously and suggested that the NF-KB-mediated inflammation is activated. The results of IPA revealed that NRF2-mediated oxidative stress response is activated in response to the oxidative stress and the progresses of gene expression, cell proliferation, cell differentiation and cell cycle are increased. The conclusion is that after treated with oleic acid, although the balance between glycometabolism and lipid metab

关 键 词：蛋白质组学 转录因子 HepG2细胞 油酸 脂肪变性 

分 类 号：Q-33[生物学]