| Purpose:The present study aimed to gain the insight into the trichloroethylene(TCE)-induced effect on the differential expression of subcellular proteins in human hepatic L-02 cells.Method:A Cell Counting Kit-8(CCK-8) assay was used to investigate the effect of TCE on the viability of L-02 cells. In our previous study, we generated a concentration-response curve based on the cell viability and figured out an IC50 value(approximately 16.0 mmol/L) for 24 h-treatment of TCE. We therefore chose 1/2 IC50 value as the maximum exposure concentration for our study. To verify the efficiency of membrane protein extraction and nuclear protein extraction, Anti-alpha 1 sodium potassium ATPase and Lamin-B1 was selected as plasma membrane marker and nucleus marker in western blot analysis, respectively.The membrane proteins and nuclear proteins of TCE-treated group and controls were extracted by Proteo Extract subcellular proteome extraction kit, respectively. The TCE-induced differentially expressions were analyzed by a two-dimensional fluorescence difference gel electrophoresis(2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight spectrometry(MALDI-TOF-MS). DAVID database was used to analyze the biological process of differential expressed proteins. STRING database was used to determine the protein-protein interactions。The differential expression of ATP synthase, β subunit(ATP5B), prolyl 4-hydroxylase, beta polypeptide(P4HB), heterogeneous nuclear ribonucleoprotein H2(RHNNPH2) and far upstream element-binding protein 1(FUBP1) were verified by Western Blot analysis in TCE-treated L-02 cells.Results:After TCE treatment for 24 h in L-02 cells, a total of 39 differentially expression proteins were identified in TCE-treated cells compared with controls. Among this, 14 of 15 in membrane fractions are suggested as membrane-associated by their transmembrane domain or subcellular location and 22 of 24 in nuclear fractions are suggested as nuclear-associated by their subcellular location. The DAVID analysis showed that the most dominant biological process that the identified proteins involved is RNA processing, and differential expressed proteins mainly participate in the process of RNA splicing. The enrichment of membrane proteins is incompact and nuclear proteins is compact by STRING analysis. The expression of ATP synthase subunit beta(ATP5B), prolyl 4-hydroxylase, beta polypeptide(P4HB), Heterogeneous nuclear ribonucleoprotein H2(HNRNPH2) and Far upsteam element-binding protein 1(FUBP1) were measured under TCE treatment by Western Blot analysis, which are consistent with the results in proteomics.Conclusion:In this study, we performed the extraction of subcellular organelles, and this method can effectively enrich the component of membrane proteins and nuclear proteins in L-02 cells. The identification and analysis of the differentially expressed subcellular proteins was achieved in combination with two-dimensional fluorescence difference gel electrophoresis(2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry(MALDI-TOF/TOF-MS). This provide new ideas and new methods that the effect of exogenous compounds to cells was studied by subcellular proteomics.TCE exposure can induce the expressed level of membrane proteins in L-02. The enrichment of differentially expressed proteins mainly appears in the process of regulation of cell death, regulation of apoptosis and cellular homeostasis.The analysis of interaction showed the enrichment of function was relatively loose. The results of Western Blot demonstrated that the alterations of ATP5 B andP4HB was consistent with proteomics.TCE exposure can induce the expressed level of nuclear proteins in L-02. The enrichment of differentially expressed proteins mainly appears in the process of RNA splicing.The analysis of interaction showed the enrichment of function was relatively compact. The results of Western Blot demonstrated that the alterations of HNRNPH2 and FUBP1 was consistent with proteomics. |
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