Proteome-wide Screening Of Transcription Factor DNA Binding Activity In HepG2 Cells After Oleic Acid Treatment

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 2mM 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 (catTFRE). By combination of catTFRE and an in-depth analysis of proteomics 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 NFκB1 is up-regulated obviously and suggested that the NFκB-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 metabolism is broken, inflammation is activated and oxidative damage is caused, HepG2 cells tend to take actions like increasing gene expression, cell proliferation and activating oxidative stress response at the transcription level and finaly keep cells survival.The results about the analysis of WCE(Whole Cell Extraction) of HepG2 cells show the same tendency. The 1677 up-regulated and 1106 down-regulated protein show the activation of cell cycle, gene expression and NRF2-mediated oxidative stress response and the inhibition of energy metabolism.To sum up, all the results show the effect of oleic acid on HepG2 cells is promote the biological process to be alive.