Hepatic stellate cells (HSCs) are the main target of radiation damage and primarily contribute to the development of radiation-induced liver fibrosis. However, the molecular events underlying the radiation-induced activation of HSCs are not fully elucidated. In the present study, human HSC line LX2 was treated with X-ray irradiation and/or TGF-β1, and profibrogenic molecules were evaluated. The iTRAQ LC-MS/MS technology was performed to identify global protein expression profiles in LX2 following exposure to different stimuli. Irradiation or TGF-β1 alone increased expression of α-SMA, collagen 1, CTGF, PAI-1, and fibronectin. Irradiation and TGF-β1 cooperatively induced expression of these profibrotic markers. In total, 102, 137, 155 dysregulated proteins were identified in LX2 cell samples affected by irradiation, TGF-β1, or cotreatment, respectively. Bioinformatic analyses showed that the three differentially expressed protein sets were commonly associated with cell cycle and protein processing in endoplasmic reticulum. The expression of a set of proteins was properly validated: CDC20, PRC1, KIF20A, CCNB1, SHCBP, TACC3 were upregulated upon irradiation or irradiation and TGF-β1 costimulation, whereas SPARC and THBS1 were elevated by TGF-β1 or TGF-β1 plus irradiation treatment. Furthermore, CDC20 inhibition suppressed expression of profibrotic markers in irradiated and TGF-β1-stimulated LX2 cells. Detailed data on potential molecular mechanisms causing the radiation-induced HSC activation presented here would be instrumental in developing radiotherapy strategies that minimize radiation-induced liver fibrosis.
Keywords: hepatic fibrosis; hepatic stellate cell; iTRAQ; irradiation; proteomics.