Proteomic identification of cyclophilin A as a potential biomarker and therapeutic target in oral submucous fibrosis

Abstract

Oral submucous fibrosis (OSF) is a pre-cancerous lesion, which is characterized by fibrosis of the oral submucosa. Despite large body of studies focusing on this disease, the molecular mechanisms underlying the progression of OSF remained unclear. In this study, 2-DE-based proteomic approaches were employed to identify the differently expressed proteins between OSF and normal tissues. In total, 88 proteins were identified with altered expression levels, including CypA. Upregulation of CypA was further validated through immunohistochemistry staining combined with Q-PCR and western blot by using clinical samples. Statistical analyses reveal that CypA expression level is correlated to the progression of OSF. Finally, functional study reveals a pro-proliferative property of CypA in fibroblast cells by using multiple in vitro models. The present data suggest that CypA might be a potential biomarker and therapeutic target for OSF, and will lead to a better understanding of OSF pathogenesis.

Keywords: CypA; fibroblast; oral submucous fibrosis; proteomics.

Figure 1. Proteomic identification of differently expressed proteins between normal and OSF tissue

A. Representative 2-DE gel images of human normal and OSF tissues. The protein extracts were separated on the pH 3-10 nonlinear IPG strips and followed with 12% SDS-PAGE in the second dimension, then visualized by CBB staining. 2-D gel images were analyzed by PDQuest software and the protein spots were labeled with numbers. B. Representative spots with changed density. C. Protein cluster analysis was performed by using Cluster software. Expression of proteins in the normal tissues was constantly set as 0. Meanwhile proteins up-regulated and down-regulated in the OSF tissue are in red and green, respectively. The intensity of the color red or green is correspondent to the level of differentiation according to the color strip. D. 88 identified proteins were classified into six groups according to their function.

Figure 2. Bioinformatics analysis of the identified proteins

A. The general workflow of bioinformatics analysis. B. Total PPI network which was generated based on the 88 identified proteins. C. The predicted CypA-associated proteins involved in apoptosis. D. The predicted CypA associated proteins involved in cell proliferation.

Figure 3. Expression of CypA is up-regulated in OSF

A. Expression of CYPA in OSF and normal tissues was examined by Q-PCR. The total mRNA was normalized by β-actin. B. Expression of CYPA in OSF and normal tissues was examined by western blot. β-actin was used as an internal control. All data were representative of at least three independent experiments. ***, P<0.001; **, P<0.01; *, P<0.05.

Figure 4. CYPA expression is associated with OSF progression

A. Collagen deposition in OSF tissues at different stages was examined by Masson staining. 20 cases of normal tissues, 20 cases of early stage OSF tissues, 20 cases of mid stage OSF tissues and 20 cases of late stage OSF tissues were used. Scale bar: left panels, 500 μm; mid panels, 200μm; right panels, 100 μm. B. Expression of CYPA in OSF tissues at different stages was examined by IHC staining. 20 cases of normal tissues, 20 cases of early stage OSF tissues, 20 cases of mid stage OSF tissues and 20 cases of late stage OSF tissues were used. Scale bar: left panels, 100 μm; mid panels, 500 μm; right panels, 100 μm. C. Expression of CYPA in OSF tissues at different stages was scored and compared. D. Expression of Annexin A2 in OSF tissues at different stages was examined by IHC staining. 15 cases of normal tissues, 15 cases of early stage OSF tissues, 15 cases of mid stage OSF tissues and 15 cases of late stage OSF tissues were used. Scale bar: left panels, 100 μm; mid panels, 500 μm; right panels, 100 μm. E. Expression of Annexin A2 in OSF tissues at different stages was scored and compared.

Figure 5. CypA is up-regulated in OSCC arising from OSF

A. 44 OSCC tissues with its surrounding OSF tissues were collected. Expression of CYPA in both OSF and OSCC areas was examined by IHC staining. Scale bar: left panels, 2 mm; mid panels, 500 μm; right panels, 100 μm. B. Expression of CYPA in either OSF or OSCC areas were scored and compared. C. Mann-Whitney was used to analyze the potential correlations between the CypA expression level and malignant transformation of OSF. D. Odds ratio analysis was used to analyze the risks of malignant transformation of OSF with CypA expression level.

Figure 6. Loss of CypA inhibits fibroblast cell proliferation

A. IMR90 and primary oral fibroblast were transfected with three distinct CypA siRNAs. Expression of CypA was examined by Q-PCR and western blot. B. IMR90 and primary oral fibroblast were transfected with siCypA1, siCypA2 or siNC, respectively. Proliferation of IMR90 and primary oral fibroblast cells was examined by CCK8 assay. C. IMR90 and primary oral fibroblast were transfected with siCypA1, siCypA2 or siNC, respectively. Proliferation of IMR90 and primary oral fibroblast cells was examined by BrdU assay. All data were representative of at least three independent experiments. ***, P<0.001; **, P<0.01; *, P<0.05.

Figure 7. Loss of CypA induces apoptosis in fibroblast cells

A. IMR90 and primary oral fibroblast were transfected with siCypA1, siCypA2 or siNC, respectively. Cell death of IMR90 and primary oral fibroblast cells was examined by PI staining. B. IMR90 and primary oral fibroblast were transfected with siCypA1, siCypA2 or siNC, respectively. Cell death of IMR90 and primary oral fibroblast cells was examined by PI/Annexin V staining-based flow cytometry assay. C. IMR90 and primary oral fibroblast were transfected with siCypA1, siCypA2 or siNC, respectively. Cell death of IMR90 and primary oral fibroblast cells was examined by TUNEL assay. All data were representative of at least three independent experiments. ***, P<0.001; **, P<0.01; *, P<0.05.

Figure 8. Loss of CypA activates AKT, ERK and Caspase 3, and reduces the mitochondrial membrane potential

A. IMR90 and primary oral fibroblast were transfected with transfected siCypA1, siCypA2 or siNC, phosphorylation of AKT and ERK was examined by western blot. B. IMR90 and primary oral fibroblast were transfected with transfected mock or CypA expression vector, phosphorylation of AKT and ERK was examined by western blot. C. IMR90 and primary oral fibroblast were transfected with transfected siCypA1, siCypA2 or siNC, activation of Caspase 3 was examined by western blot. D. IMR90 and primary oral fibroblast were transfected with transfected siCypA1, siCypA2 or siNC, mitochondrial membrane potential was examined by JC-1 staining. All data were representative of at least three independent experiments. ***, P<0.001; **, P<0.01; *, P<0.05.