SET-mediated Self-regulation Mechanisms Of Nucleolin In TCE-induced Hepatic Cytotoxicity

Background and objectivesTrichloroethylene (TCE) was a low cost and wildly used chemical in many industries. For instance, it was used as a solvent for a variety of organic materials, degreasing of metal, adhesive paint, component of anaesthetics and polyvinyl chloride production. TCE was also used for plastic productions, such as pipe lines, wires and packing materials. Recent years, TCE has become one of the most dangerous occupational toxicants in Guangdong province of China. It has brought occupational hazard and economic damages to Shenzhen in the past few years. TCE could be absorbed into human body by reposiry tract system and skin resorption. Nowadays, TCE is being as an enviromental toxicant rather than an industrial material. In vitro and in vivo studies showed that TCE could be genetic toxic, carcinogenic and mutagenesis. Based on plenting supporting evidence, the U.S. Environmental Protection Agency (EPA) released a final health assessment for TCE characterizing it as a human carcinogen in 2011. Less than a year, International Agency for Research on Cancer (IARC) has also identified TCE as a carcinogenic hazard. Chances of occupational exposure to TCE has been kept reducing because of alternative usages of TCE and occurrence of alternative compounds. However, TCE has turned into a serious enviromental issue by becoming one of the main ground water contaminations. Thus the risks for exposure to TCE is becoming even higher in our daily lives. Liver is one of the major organs in which TCE was metabolised and attacked. One metabolism pathway of TCE was was oxidized into the intermediate of trichloroethylene epoxide by cytochrome P450 (CYP4500). The products with lower toxicity, such as chlorinated aldehyde, trichloroethanol and trichloroacetic acid were then excreted into the urine. Another pathway was becoming S-(1,2-two vinyl chloride) glutathione (DCVG) by binding to the glutathione (GSH) under the effect of glutathione S-transferase (GST). Then DCVG was metabolized into S-(1,2-two vinyl chloride)-L-cysteine (DCVC). However, the details of TCE-induced liver damage was still unclear and needed a full and systematic study.Our previous study showed TCE-induced hepatic cytotoxicity in HL-7702 (L-02) cells. We screened the TCE-induced differentially expressed proteins in L-02 cells by using 2D electrophersis for. The results showed that SET protein was associated with TCE by dose-effect relationship. We further analyzed the role of SET by constructing the SET-siRNA stable transfected L-02 cells. Defeciency of SET protein in L-02 cells could alleviate the TCE-induced cytotoxiciy, thus SET may mediated the TCE-induced hepatic cytotoxicity in L-02 cells. SET protein was involved in many important biological processes, including cell apoptosis, nucleosome assembly and chromatin remodeling, it also functioned as a molecular chaperon of histones. SET protein was a part of the inhibition complex of histone acetyl transferase which controlled the deacetylation process. Many reports provided the evidences of SET as a oncoprotein, it was involved in tumor proliferation and progression. Although SET protein was an endogenous and specific inhibitor of phosphotase 2A (PP2A). it could also activate phosphotase 1 (PP1) in the presence of 2+ manganese ions. Thus SET played a key role in the process of protein phosphorylation.Protein phosphorylation is the basic way of molecular regulation and signal transduction in eukaryotic cells. The protein kinase transfers the phosphate group from adenosine triphosphate (ATP) to the specific amino acid residues in the target protein. Some kind of protein functions could be activated or inhibited by phosphorylation modifications and further alter the process of signal transduction, metabolism, apoptosis, cell differentiation and so on.Although SET protein has been known to paly an imoportant role in protein phosphorylation, the mechanisms of how it involved in the modification process is still unclear and waiting to be explored. In this study we further analyzed SET-mediated phosphorylation alterations in TCE-induced hepatic cytotoxicity in L-02 cells. We used phosphoproteomics technics to screen for TCE-induced and SET-mediated alterations of protein phosphorylation, then we studied SET-mediated nucleolin overall phosphoryaltion and self-regulation process.MethodsPhosphoproteomics study of TCE-induced hepatic cytotoxicity in L-02 cells1. Cell culture and treatmentL-02 cells and SET-siRNA transfected L-02 cells were cultured in RPMI-1640 medium supplemented with 12% fetal bovine serum (GIBCO),100 units/ml penicillin and 100 μg/ml streptomycin in 37 ℃ with 5% CO2. The cells were treated with 8 mmol/L TCE for 24 h without fetal bovine serum.2. Protein extraction and digestionImmediately after the treatment, the cells were collected, washed, and then lysed on ice with PhosphoSafeTM Extraction Reagent. Quantification of the proteins was performed following the manufacturer’s instructions for the 2D Quant Kit.100 μg of total protein in each sample was used for reduction and alkylation and then digested by 1 μg trypsin.3. iTRAQ labeling, phosphopeptide enrichment and sample desaltingThe digestion products were lyophilized and then re-suspended in 100 mmol/L TEAB. iTRAQ reagents (113,114 115 and 116 were used to label samples and 121 was used to label internal standard) were prepared and labeled to each sample following the manufacturer instructions. TiO2 based IMAC technology was applied to enrich the phosphopeptides. The enriched peptides were desalted by using C18 spin columns.4. LC-MS/MS analysisSamples were re-dissolved in 2% acetonitrile,0.1% formic acid, and loaded on a trap column. Then, an elution gradient of 5-35% acetonitrile (0.1% formic acid) in a 90 min gradient was used on an analytical column. Peptides were ionized and atomized into mass spectrometry by a Nanospray Ⅲ source. Precursor ions and product ions were extracted by using ProteinPilot Software v.4.5 and protein searching was conducted by using Mascot, the randomized database was used to control the false positive rate (FDR) under 1%.5. Data quantification and bioinformatics analysisImpurities correction and data normalization were processed by ProteinPilot Software v.4.5, and the internal standard was chosen as dominator. Phosphopeptides only appeared in all three replicates were preserved to quantify. The differential phosphoproteins were enriched by R package clusterProfiler with KEGG pathways and GO categories (biological process, molecular function and cellular component).6. Validation of differential protein phopshorylation by Western-blot analysisThe proteins were extracted electrophersed, then transferred into PVDF membranes. Antibodies were diluted in TBST buffer and incubated at room temperature for 60 min. Protein bands were visualized using ECL substrate and an ImageQuant RT ECL System (GE Healthcare). The relative quantification of the proteins was performed using ImageQuant TL-ID Analysis Tool.SET-mediated self-regulation of nucleolin in TCE-induced L-02 cytotoxicity1. Overall phosphorylation and exression of nucleolin analysis by Western-blotThe proteins were extracted by PhosphoSafe Extraction Reagent. The SDS-PAGE was prepared with Phos-tag reagent. After electrophersis, the proteins were then transferred into PVDF membranes. Antibodies were diluted in TBST and incubated for 60 min at room temperature. Protein bands were visualized using ECL substrate and an ImageQuant RT ECL System (GE Healthcare). The relative quantification of the proteins was performed using ImageQuant TL-1D Analysis Tool.2. C-myc inhibition in L-02 cells and expression analysis of nucleolin by Western-blotL-02 cells were cultured in six well plates at 37 ℃,5% CO2. and treated with different concentrations of c-myc inhibitor (0 μmmol/L、20μmmol、40μmmol、80 μmmol、100 μmmol) for 24 hours. The cells were then collected for detecting the expression of c-myc and nucleolin by Western-blot analysis.3. SET-mediated interaction between nucleolin and c-myc in TCE-induced L-02 cytotoxicityL-02 cells and SET-siRNA transfected L-02 cells were treated with 8 mmol/L TCE for 24 hours, then collected for protein extraction and quantification. The nucleolin and c-myc antibodies were cross-linked with A/G Plus agrose beads. Nucleolin and c-myc were applied as bait protein respectively in the Co-IP experiments. Western-blot analysis was used to detect the level of target proteins which were pulled down by bait proteins.SET-mediated transcriptome study in TCE-induced L-02 cytotoxicity1. Cell treatment, fluorescence labeling and probe hybridizationL-02 cells and SET-siRNA transfected L-02 cells were treated with 8 mmol/L TCE for 24 hours respectively, then the total RNA was extracted by chloroform/Trizol mixture. Isopropyl alcohol and anhydrous ethanol to remove salts, proteins and DNA moleculars and precipitate RNA. Sample labeling with fluorescence dyes and hybridize with the probes on the microarray chips was done by following the instructions of Eukaryotic Poly-A RNA Control Kit, Eukaryotic Hybridization Control Kit and Hybridization, Wash, and Stain Kit. The microarray chips were scanned by GeneChip(?) Scanner 3000 and images were processed by AGCC software.2. Quality control, relative quantification and gene set variation analysisQuality control for microarray study was conducted by using the R package Affy. The outlier detection and RNA degragation test were included in the accessment of data quality. Before the quantification process, gene signals were calculated as median of different probes, and sum of different transcripts. For further analysis of SET-mediated alterations of pathways, GSVA algorithm was applied to screen the activated/inactivated pathways.ResultsPhosphoproteomics study of TCE-induced hepatic cytotoxicity in L-02 cells1.107 phosphorylation proteins and 1878 phosphorylation sites were identified. Among the 1878 phosphorylation sites,57 sites were recorded in the PhosphoSitePlus database, and 174 sites were recorded in the Phospho.ELM database.2. 44 phosphorylation sites of 37 phosphorylation proteins were found abnormally (de)-phosphorylated with statistical significance in L-02 cells and SET-siRNA transfected L-02 cells treated with TCE respectively.3. SET-mediated differentially phosphorylated at 13 sites of 14 proteins in TCE-induced hepatic cytotoxicity.4. Functional enrichment analysis showed that these phosphoproteins were related to 5 KEGG pathways,33 molecular functions, biological processes and 51 cellular components.5. Abnormally phosphorylated at 4E-BP1 (T46) (2.52±0.23、2.65±0.44、1.35± 0.21, P=1.268e-10) and MCM2(S139) (0.34±0.07、0.75±0.08、4.50±1.07, P=5.714e-11) were validated by Western-blot analysis.SET-mediated self-regulation of nucleolin in TCE-induced L-02 cytotoxicity1. The overall phosphorylation of nucleolin decreased in L-02 cells treated with TCE and increased in SET-siRNA transfected L-02 cells treated with TCE. The alteration pattern of nucleolin expression was reversed compared to the alteration pattern of nucleolin overall phosphorylation.2. The expression of nucleolin was down-regulated along with the inhibition of c-myc.3. The results of Co-IP analysis showed that the binding between c-myc and nucleolin became weaker in L-02 cells treated with TCE and stronger in SET-siRNA transfected L-02 cells treated with TCE.SET-mediated transcriptome study in TCE-induced L-02 cytotoxicity1. Quality accessment showed that afer log-transformation, there was no overall signals of microarrays could be detected as outliers. RNA degragation test showed that no significant RNA degragation could be observed.2. 4 genes were both differentially expressed in L-02 cells and SET-siRNA transfected L-02 cells treated with TCE.39 genes were found abnormally expressed only in SET-siRNA transfected L-02 cells treated with TCE. Thus the transcription of these genes could be SET-mediated in L-02 cells treated with TCE.3. Of all the SET-mediated differentially expressed genes, three apoptosis-related genes, YWHAQ, FCER1G and CNTFR were found being associated with c-myc and nucleolin by STRING analysis.4. Gene set variation analysis showed that 13 pathways were inactivated and 7 pathways were activated in L-02 cells treated with TCE.5 pathways were inactivated and 3 pathways were activated in L-02 cells transfected with SET-siRNA. Thrombin-Par4 pathway was activated in SET-siRNA transfected L-02 cells treated with TCE.Conclusion1.107 phosphorylation proteins and 1878 phosphorylation sites were identified.44 phosphorylation sites of 37 phosphorylation proteins were found abnormally (de)-phosphorylated in different groups. SET-mediated differentially phosphorylated at 13 sites of 14 proteins in TCE-induced hepatic cytotoxicity. Two phosphorylation sites of two proteins were validated by Western-blot analysis.2. Based on prevous observations, we found that SET-mediated overall phosphorylation alteration of nucleolin, which could impact its binding to c-myc and lead to expression changes in TCE-induced hepatic cytotoxicity.3. SET-mediated abnormally transcription of multiple genes were identified in TCE-induced hepatic cytotoxicity. Three apoptosis-related genes, YWHAQ、 FCER1G and CNTFR were found being associated with c-myc and nucleolin. The observations suggested that the transcription of these genes may be mediated by SET through regulation of nucleolin.