The integrated method of LC-MS/MS and TMT labeling was used to analyze the proteomic changes in the PM2.5-exposed HBE cells and c-fos silenced HBE cells. The DEPs with post-translational modifications were extracted from the UniprotKB database, and the differential proteins were obtained according to the criteria of P < 0.05. The volcano diagram in Figure 1 shows the differential protein expression profile, and the heat map in Figure 1 shows the different distribution of DEPs in the c-fos silenced HBE group between the experimental group and the control group. According to the criteria of FC ≤ 0.83 or FC ≥ 1.2 and P value < 0.05, top 15 differential proteins in the HBE group were selected, of which 6 were up-regulated and 9 down-regulated. Among c-fos silenced HBE groups, 4 proteins were up-regulated and 11 were down-regulated in top 15 DEPs (Tables 1-2).
Figure 1. Volcano gram and heat map of proteomics analysis. (A) Volcanic map of PM2.5-induced DEPs in HBE cells. (B) Volcanic map of PM2.5-induced DEPs in c-fos silenced HBE cells. (C) Heat map of PM2.5-induced DEPs in HBE cells. (D) Heat map of PM2.5-induced DEPs in c-fos silenced HBE cells.
Protein accession number Protein name Gene FC Up/down P value Q9NQ84-2 G-protein coupled receptor family C group 5 member C GPRC5C 1.30 up 0.01 Q92597 Protein NDRG1 NDRG1 1.27 up < 0.01 O75874 Isocitrate dehydrogenase (NADP) cytoplasmic IDH1 1.25 up 0.02 O95749 Geranylgeranyl pyrophosphate synthase GGPS1 1.25 up 0.01 P14324 Farnesyl pyrophosphate synthase FDPS 1.25 up < 0.01 P82930 28S ribosomal protein S34, mitochondrial MRPS34 1.22 up < 0.01 P02768 Serum albumin ALB 0.57 down < 0.01 P02765 Alpha-2-HS-glycoprotein AHSG 0.59 down 0.01 Q8N257 Histone H2B type 3-B HIST3H2BB 0.67 down 0.01 Q15004 PCNA-associated factor PCLAF 0.68 down 0.01 P02774-3 Vitamin D-binding protein GC 0.74 down 0.02 Q8NI35 InaD-like protein PATJ 0.75 down 0.04 Q8N2C7 Protein unc-80 homolog UNC80 0.76 down 0.01 P35237 Serpin B6 SERPINB6 0.77 down 0.01 P13693 Translationally-controlled tumor protein TPT1 0.81 down < 0.01
Table 1. Top 15 differentially expressed proteins in the HBE group
Protein accession number Protein name Gene FC Up/down P value Q9UBT3 Dickkopf-related protein 4 DKK4 1.29 up 0.01 P53602 Diphosphomevalonate decarboxylase MVD 1.24 up 0.04 Q92820 Gamma-glutamyl hydrolase GGH 1.22 up < 0.01 O95471 Claudin-7 CLDN7 1.22 up 0.01 Q9Y6V0-5 Protein piccolo PCLO 0.55 down < 0.01 P02768 Serum albumin ALB 0.57 down < 0.01 P05090 Apolipoprotein D APOD 0.58 down < 0.01 Q15004 PCNA-associated factor PCLAF 0.60 down < 0.01 P02765 Alpha-2-HS-glycoprotein AHSG 0.61 down < 0.01 P02787 Serotransferrin TF 0.68 down 0.05 Q8N2C7 Protein unc-80 homolog UNC80 0.69 down < 0.01 Q8NI35 InaD-like protein PATJ 0.71 down 0.01 P02788 Lactotransferrin LTF 0.72 down 0.02 P35237 Serpin B6 SERPINB6 0.75 down < 0.01 O00762 Ubiquitin-conjugating enzyme E2 C UBE2C 0.76 down < 0.01
Table 2. Top 15 differentially expressed proteins in the c-fos silenced HBE group
Normal HBE cells and c-fos silenced HBE cells were exposed to PM2.5 treatment. The analysis of GO and KEGG annotations showed that HBE group and c-fos silenced HBE group were involved together in the biological processes in which the differential proteins were mainly related with mRNA splicing, via spliceosome, cell–cell adhesion, and translational initiation. The differential proteins in the HBE group are mainly involved in biological processes such as translational elongation, regulation of cellular response to heat, and negative regulation of apoptotic process. The c-fos silenced HBE group is mainly involved in biological processes such as ribosomal small subunit biogenesis, regulation of translational initiation, and cell division. The cellular components of differential proteins in two groups are mainly distributed in extracellular exosome, mitochondrion, ribosome, myelin sheath, and other components. The cellular components of differential proteins in the HBE group were mainly distributed in the spliceosomal complex, catalytic step 2 spliceosome, and lysosomal lumen, whereas in the c-fos silenced HBE group, the differential proteins involved were mainly in the mitochondrial inner membrane and perinuclear region of cytoplasm. The molecular functions of the differential proteins in two groups are mainly involved in poly (A) RNA binding, protein binding, cadherin binding in cell–cell adhesion, and structural constituent of ribosome. The molecular functions of the differential proteins in HBE group are mainly associated with ubiquitin protein ligase binding, Arp2/3 complex binding, protein kinase binding, and G-protein coupled receptor binding. The molecular functions of the differential proteins in c-fos silenced HBE group were mainly related to translation initiation factor activity, mRNA 3'-UTR binding, and ribosome binding (Figures 2-4).
Figure 2. Biological processes of differentially expressed proteins after PM2.5 exposure. (A) HBE group. (B) c-fos silenced HBE group.
Figure 3. The cell components of differentially expressed proteins after PM2.5 exposure. (A) HBE group. (B) c-fos silenced HBE group.
KEGG analysis showed that the pathways in which the DEPs in two groups participate together are carbon metabolism, ribosome, citrate cycle (TCA cycle), amino acid biosynthesis, and spliceosome. Glycolysis/gluconeogenesis is the only pathway involved in differential proteins in the HBE group. The pathways of differential protein participation in c-fos silenced HBE group include protein processing in endoplasmic reticulum, aminoacyl-tRNA biosynthesis, pyruvate metabolism, lysosome, valine and other pathways (Figure 5).
The STRING database was used to analyze the protein–protein interactions of the screened DEPs. The results were visualized in cytoscape shown in Figure 3A. The Hub network in the two network diagrams is obtained using the cytoHubba plug-in in cytoscape, which is the key protein in the protein interaction network. The Hub proteins in the normal HBE group are HNRNPA2B1, HNRNPA3, RBMX, HNRNPU, SNRPA, HNRNPF, SF3B1, SF3A3, HNRNPL, and ALYREF. In the c-fos silenced HBE group, the Hub proteins were RPS9, RPS21, RPS7, RPS25, RPL31, RPS27A, RPS15, RPS15A, RPS28, and RPL30 (Figure 6, Table 3).
Group Abbreviation of protein Protein name FC Up/down HBE group vs. HBE + PM2.5 group HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins A2/B1 1.10 up HNRNPA3 Heterogeneous nuclear ribonucleoprotein A3 1.09 up RBMX RNA-binding motif protein, X chromosome 1.09 up HNRNPU Heterogeneous nuclear ribonucleoprotein U 1.07 up SNRPA U1 small nuclear ribonucleoprotein A 1.07 up HNRNPF Heterogeneous nuclear ribonucleoprotein F 1.06 up SF3B1 Splicing factor 3B subunit 1 1.06 up SF3A3 Splicing factor 3A subunit 3 1.06 up HNRNPL Heterogeneous nuclear ribonucleoprotein L 1.04 up ALYREF THO complex subunit 4 0.83 down c-fos silenced group vs. c-fos silenced + PM2.5 group RPS9 40S ribosomal protein S9 0.87 down RPS21 40S ribosomal protein S21 0.89 down RPS7 40S ribosomal protein S7 0.90 down RPS25 40S ribosomal protein S25 0.92 down RPL31 60S ribosomal protein L31 0.92 down RPS27A Ubiquitin-40S ribosomal protein S27a 0.93 down RPS15 40S ribosomal protein S15 0.93 down RPS15A 40S ribosomal protein S15a 0.94 down RPS28 40S ribosomal protein S28 0.94 down RPL30 60S ribosomal protein L30 0.98 down
Table 3. Hub differential proteins in the HBE group and c-fos-silenced HBE group
Figure 6. Network diagram of differential protein interactions after PM2.5 exposure. (A) Interaction network diagram of differential proteins in HBE group; (B) Interaction network diagram of differential proteins in c-fos silent HBE group; (C) Hub protein network diagram of HBE group; (D) c-fos Hub protein network diagram of HBE group.
Proteomics Study on the Differentially Expressed Proteins in c-fos-silenced Cells Exposed to PM2.5
- Received Date: 2020-03-02
- Accepted Date: 2020-07-02
|Citation:||CAI Ying, ZHENG Kai, LI Run Bing, YU Shu Yuan, LIU Ning, JI Jia Jia, YANG Chen, WU De Sheng, QIN Shuang Jian, LI Bo Ru, ZHANG Zhao Hui, XU Xin Yun. Proteomics Study on the Differentially Expressed Proteins in c-fos-silenced Cells Exposed to PM2.5[J]. Biomedical and Environmental Sciences, 2020, 33(9): 680-689. doi: 10.3967/bes2020.089|