Integrative analysis of proteomic and transcriptomic data for identification of pathways related to simvastatin‐induced hepatotoxicity
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Hepatocytes are used widely as a cell model for investigation of xenobiotic metabolism and the toxic mechanism of drugs. Simvastatin is the first statin drug used extensively in clinical practice for control of elevated cholesterol or hypercholesterolemia. However, it has also been reported to cause adverse effects in liver due to cellular damage. In this study, for proteomic and transcriptomic analysis, rat primary hepatocytes were exposed to simvastatin at IC20 concentration for 24 h. Among a total of 607 differentially expressed proteins, 61 upregulated and 29 downregulated proteins have been identified in the simvastatin‐treated group. At the mRNA level, results of transcriptomic analysis revealed 206 upregulated and 41 downregulated genes in the simvastatin‐treated group. Based on results of transcriptomic and proteomic analysis, NRF2‐mediated oxidative stress response, xenobiotics by metabolism of cytochrome P450, fatty acid metabolism, bile metabolism, and urea cycle and inflammation metabolism pathways were focused using IPA software. Genes (FASN, UGT2B, ALDH1A1, CYP1A2, GSTA2, HAP90, IL‐6, IL‐1, FABP4, and ABC11) and proteins (FASN, CYP2D1, UG2TB, ALDH1A1, GSTA2, HSP90, FABP4, and ABCB11) related to several important pathways were confirmed by real‐time PCR andWestern blot analysis, respectively. This study will provide new insight into the potential toxic pathways induced by simvastatin.
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Figure S1. E.coli Protein and peptide numbers for the 1D and 2D‐3 fraction E.coli experiments.
Figure S2. The Venn diagram of the overlap of E.coli proteins and peptides numbers from 1D‐LC (A) and 2D‐LC (B) analysis between two biological replicates, respectively.
Figure S3. Comparison of subcellular localization from proteins identified by gel‐assisted digestion and in‐solution digestion, respectively.
Figure S4. Treatment with simvastatin resulted in dose‐dependent diminishment of viability of rat primary cultured hepatocytes (A).
Figure S5. The numbers of proteins and peptides identified from 1D‐LC and 2D‐LC methods (A), the Venn diagram of the overlap of proteins identified from 1‐LC and 2D‐LC (B).
Figure S6. 2D‐5fraction control (A) and simvastatin‐treated (B) rat primary hepatocyte separation.
Figure S7. Subcellular localization (A) and functional categorization (B) of differentially expressed genes in simvastatin‐induced hepatotoxicity.
Figure S8. Correlation between protein and corresponding transcript modulation. Results presented all common proteins and genes (A) and differentially expressed proteins and genes (B).
Figure S9. A boxplot of the ranking of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs).
Figure S10. mRNA expressions of HMG‐COA reductase (HMG‐COAR), and low density lipoprotein receptor (LDLR) in simvastatin‐treated hepatocytes.
|pmic7353-sup-0002-TableS1.xls7 MB||Table S1. List of proteins identified from twelve runs of control and simvastatin‐treated groups.|
|pmic7353-sup-0003-TableS220.3 KB||Table S2. Gene specific primers used in real‐time PCR confirmation experiments.|
|pmic7353-sup-0004-TableS3.xls69 KB||Table S3. mRNA changes in simvastatin‐treated hepatocytes at p < 0.05 and fold change greater than 2.0 or less than 0.5.|
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