Study On Molecular Mechanism Of Systematic Dysfunction In Hepatoma Cells After Arginine Starvation

Cancer has become one of the major hazards of human death,and liver cancer is one of the malignant tumors having the highest fatality rate,therefore the treatment of liver cancer has become a research focus in recent years.Over 40%of tumor cells cannot synthesize arginine themselves,while arginine deiminase(ADI for short)can degrade exogenous arginine and kill tumor cells which make ADI a new potential anti-tumor drug.We take hepatoma cells and normal liver cell as object in this study.ADI is transfected in these cells respectively,the subsequent flow cytometry shows that ADI has strong killing effect on hepatoma cells while almost non-toxic effect on normal liver cell,this result gurantee the feasibility of taking ADI as treatment of liver cancer.In order to uncover the molecular mechanism of ADI effect on hepatoma cells,gene chip is used in this study to detect changes of gene expression at transcriptome level.ADI was transfected into hepatoma cells and normal liver cell respectively and total RNA was extracted,after the subsequent chip hybridization,raw data normalization,differentially expressed gene screening,five pathways of interest was obtained:apoptosis,autophagy,cell cycle,cell migration,inflammation and immunity.Real-time PCR and western blot was then used to verify the result of gene chip,in addition,mitochondrial damage detection,caspase3 and caspase9 activity change detection,confocal laser scanning microscopy,flow cytometry and other technologies was used to prove the role of ADI in these metabolic pathways.Furthermore,in order to find the protein that interacts with ADI in cells,yeast two-hybrid was used and we obtain three candidate proteins,the most int erested ferritin light chain(FTL for short)was selected for the subsequent co-i mmunoprecipitation and immunofluorescence co-localization test,and the interac tion between ADI and FTL was proved eventually.