Protein Expression Profile in Rat Silicosis Model Reveals Upregulation of PTPN2 and Its Inhibitory Effect on Epithelial-Mesenchymal Transition by Dephosphorylation of STAT3
Abstract
:1. Introduction
2. Results
2.1. Protein Expression Profiles Undersilica Exposure
2.2. Gene Ontology, KEGG Pathway Analysis and Protein-Protein Network Analysis of Differentially Abundant Proteins
2.3. Validation of iTRAQ-Screened Proteins Exhibiting Increased Expression Levels in the Different Experimental Groups of Silicotic Rats
2.4. Validation of iTRAQ-Screened Proteins Exhibiting Increased Expression Levels in SiO2-Stimulated MLE-12 Cells
2.5. PTPN2 Overexpression Inhibited the SiO2-Stimulated Epithelial-Mesenchymal Transition of MLE-12 Cells by Dephosphorylating STAT3
2.6. PTPN2-Gene Silencing Promoted the SiO2-Stimulated EMT of MLE-12 Cells by Phosphorylating STAT3
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. Rat Models
4.3. Protein Extraction, Digestion, and iTRAQ Labeling
4.4. Separation of Peptides and LC Mass Spectrometric Analysis
4.5. Bioinformatic Analysis
4.6. Cell Culture and Treatment
4.7. Design and Construction of Lentivirus Vector
4.7.1. Construction of Plasmid Vector for PTPN2 Gene Knockdown Lentivirus
4.7.2. Construction of Plasmid Vector for PTPN2 Gene Overexpress Lentivirus
4.7.3. The Process of Lentivirus Packaging Plasmids
4.8. Lentiviral Vector Infection
4.9. Western Blot Analysis
4.10. Immunofluorescence Staining
4.11. Immunohistochemistry Staining
4.12. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Ethics Approval
References
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No. | Protein Name | Accession Number | Fold Change |
---|---|---|---|
1 | Acidic mammalian chitinase | [CHIA_RAT] | 4.277554 |
2 | Tyrosine-protein phosphatase non-receptor type 2 | [PTN2_RAT] | 2.292036 |
3 | Protein Vrk1 | [VRK1_RAT] | 2.126730 |
4 | B-factor, properdin | [Q6MG74_RAT] | 1.970988 |
5 | Complement component 4, gene 2 | [Q6MG90_RAT] | 1.822588 |
6 | Cd68 molecule | [Q4FZY1_RAT] | 1.773507 |
7 | Protein Spcs1 | [D3ZFK5_RAT] | 1.749611 |
8 | Equilibrative nucleoside transporter 3 | [ENT3_RAT] | 1.731603 |
9 | BPI fold-containing family B member 1 | [BPIB1_RAT] | 1.716078 |
10 | Monocarboxylate transporter 4 | [MOT4_RAT] | 1.680098 |
11 | Transmembrane glycoprotein NMB | [GPNMB_RAT] | 1.676391 |
12 | A-kinase anchor protein SPHKAP | [SPKAP_RAT] | 1.672275 |
13 | Complement C4 | [CO4_RAT] | 1.652953 |
14 | Olfactory receptor | [D3ZRJ8_RAT] | 1.652572 |
15 | Phospholipase B-like 1 | [PLBL1_RAT] | 1.585530 |
16 | Chitinase 3-like 1 protein (Fragment) | [A4LA56_RAT] | 1.558782 |
17 | Cell division cycle protein 123 homolog | [CD123_RAT] | 1.546649 |
18 | Protein Stk33 | [D4A165_RAT] | 1.532094 |
19 | Beta-defensin 4 | [DEFB4_RAT] | 1.519443 |
20 | UDP-glucuronosyltransferase | [Q6T5E8_RAT] | 1.506820 |
21 | Protein Slc16a2 | [G3V9C2_RAT] | −1.877088 |
22 | Carboxylic ester hydrolase | [A0A0G2JV37_RAT] | −1.842240 |
23 | Protein Spns2 | [D3ZPS4_RAT] | −1.796205 |
24 | PDZ domain-containing protein 2 | [F1M785_RAT] | −1.666518 |
25 | Protein Dcp1b | [D3ZZJ7_RAT] | −1.571295 |
26 | CD34 antigen isoform 2 | [B1PLB2_RAT] | −1.566583 |
27 | Protein Shroom2 | [SHRM2_RAT] | −1.541996 |
28 | Periaxin | [PRAX_RAT] | −1.528930 |
Vector Number | Lenticirus Titer |
---|---|
pHS-ASR-LW265 | 1.08 × 108 TU/mL |
pHS-ASR-LW266 | 1.02 × 108 TU/mL |
pHS-ASR-LW267 | 1.37 × 108 TU/mL |
pHS-ASR-LW198 | 1.09 × 108 TU/mL |
pHS-AVC-LW1049 | 1.03 × 108 TU/mL |
pHS-BVC-LW299 | 1.83 × 108 TU/mL |
Vector Number | Inserted Content | shRNA Sequence |
---|---|---|
pHS-ASR-LW265 | PTPN2 shRNA01 | 5′-GCATTCTACGGAAACGTATTC CGAAGAATACGTTTCCGTAGAATGC-3′ |
pHS-ASR-LW266 | PTPN2 shRNA02 | 5′-GCATTAATCTCAAGGGTTTGT CGAAACAAACCCTTGAGATTAATGC-3′ |
pHS-ASR-LW267 | PTPN2 shRNA03 | 5′-GCAGGTAAGAAGGATGTTTCC CGAAGGAAACATCCTTCTTACCTGC-3′ |
pHS-ASR-LW198 | Neg control shRNA | 5′-CCTAAGGTTAAGTCGCCCTCGC CGAAGCGAGGGCGACTTAACCTTAGG-3′ |
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Zhu, Y.; Yao, J.; Duan, Y.; Xu, H.; Cheng, Q.; Gao, X.; Li, S.; Yang, F.; Liu, H.; Yuan, J. Protein Expression Profile in Rat Silicosis Model Reveals Upregulation of PTPN2 and Its Inhibitory Effect on Epithelial-Mesenchymal Transition by Dephosphorylation of STAT3. Int. J. Mol. Sci. 2020, 21, 1189. https://doi.org/10.3390/ijms21041189
Zhu Y, Yao J, Duan Y, Xu H, Cheng Q, Gao X, Li S, Yang F, Liu H, Yuan J. Protein Expression Profile in Rat Silicosis Model Reveals Upregulation of PTPN2 and Its Inhibitory Effect on Epithelial-Mesenchymal Transition by Dephosphorylation of STAT3. International Journal of Molecular Sciences. 2020; 21(4):1189. https://doi.org/10.3390/ijms21041189
Chicago/Turabian StyleZhu, Ying, Jingxin Yao, Yuxia Duan, Hong Xu, Qiyun Cheng, Xuemin Gao, Shumin Li, Fang Yang, Heliang Liu, and Juxiang Yuan. 2020. "Protein Expression Profile in Rat Silicosis Model Reveals Upregulation of PTPN2 and Its Inhibitory Effect on Epithelial-Mesenchymal Transition by Dephosphorylation of STAT3" International Journal of Molecular Sciences 21, no. 4: 1189. https://doi.org/10.3390/ijms21041189