Downregulation of Calreticulin and Annexin A2 in Cholesteatoma by 2-DE Analysis

Background: Many factors are thought to be associated with the development of cholesteatoma, while the mechanisms of its formation remain unclear. This study aimed to identify the potential mechanisms of the proliferation and growth of cholesteatoma by analysis of the differential expressions of proteins in cholesteatoma and retroauricular skin tissue collected from patients. Methods: Comparative proteomics analyses using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), in addition to immunohistochemical analysis, were conducted to identify differentially-expressed proteins in cholesteatoma tissue as compared with retroauricular skin tissue. Western blotting was also employed to verify the expression patterns of the specific proteins identified by 2-DE and to measure the changes in potential modulators related to cholesteatoma proliferation and growth. Results: Calreticulin (CRT) and annexin A2 (AnxA2) were identified as being differentially-expressed in cholesteatoma by 2-DE and LC-MS/MS, the results of which were in agreement with the results of immunohistochemical analysis and western blotting. Downregulation of CRT and AnxA2 were observed in cholesteatoma. Conclusion: Our data suggested that CRT and AnxA2 were associated with cholesteatoma. We speculated that the reduced expression of CRT and the persistent inflammatory response play important roles in the epithelial proliferation of cholesteatoma.

regarding cholesteatoma development have been proposed, enabling otologists to improve treatment. However, some processes are still unclear. Recent studies of the pathogenesis of acquired cholesteatoma that focused on epithelial hyperproliferation, altered differentiation, apoptosis mechanisms and inflammation provided more detail at the cellular and molecular levels regarding the pathogenesis of cholesteatoma (4,(6)(7)(8)(9)(10).
Several studies proposed that cholesteatoma is linked to osteoclast activation and a number of cellular responses (11)(12)(13). Multiple theories are currently acceptable models for cholesteatoma formation, but the surfeit of possible processes render the pathogenesis of cholesteatoma a molecular and cellular puzzle. Prevention of complications and the recurrence of cholesteatoma are the main issues of concern for otologists today. Identifying differentially-expressed proteins in cholesteatoma may uncover the mechanisms of cholesteatoma formation, and could therefore benefit the development of new treatments for the disease.
At present, there are still few studies on Cholesteatomas using proteomic analysis methods. Previous studies using 2D electrophoresis, MALDI-TOF/MS analysis and Immunohistochemical staining found that FKH 5-3 and titin are more abundant in congenital cholesteatoma tissue. It is speculated that congenital cholesteatoma origins may differ from those of acquired cholesteatomas, which originate from retraction pocket Epithelia (14). Randall et al. used mass-based spectrometry proteomic approach to assess relative changes between proteins found in the middle ear mucosa and postauricular skin relative to the cholesteatoma stroma. In addition, the inclusion of the middle ear mucosa increases the amount of novel protein in the matrix compared to postauricular skin. Many of the potential biomarkers found in the study were used to assess residual or recurrent disease, particularly BLMH, TYMP, FLBP5, FLG / FLG2 and CKAP4 (15).
The authors' results of extensive bioinformatics analysis of the first large-scale proteomics study of cholesteatoma. Proteomics studies were implicated suggest everal altered biological processes associated with the pathology of cholesteatoma. Down-regulation of several extracellular matrices and basement membrane proteins (such as COL18A1 and NID2) observed may have a major impact on tissue integrity. The up-regulation of ELANE and pro-inflammatory S100 proteins (eg S100A7A and S100A7) is mainly regulated biological areas in cholesteatoma (16).
In the present study, we used a comparative proteomics approach to distinguish proteins that were differentially-expressed in cholesteatoma tissue in comparison with retroauricular skin tissue obtained from patients.
In the current study, we employed 2-DE and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate proteins differentially-expressed in cholesteatoma as compared with normal retroauricular skin, and elucidated their pathophysiological significance, with the aim of identifying new targets for cholesteatoma treatment.

Sample collection
Tissue samples from five patients (one male and four females) with a mean age of 56.8 years (range, 40-65 years) were analyzed in this study (Table 1) First-dimension electrophoresis was carried out using a GE Healthcare Ettan IPGphor 3, as previously described(17). Proteins (250 ∝ g) extracted from cholesteatoma and retroauricular skin tissues were loaded on 11-cm IPG strips (pI 4-7 and pI 3-11, Immobiline DryStrip) for first-dimension electrophoresis, followed by separation on sodium dodecyl sulphate polyacrylamide electrophoresis gel (12.5%) using an SE 600 Ruby (Hoeffer) at 150 V for 6.5 h. ProteinTech Group, Chicago, IL, USA) at room temperature for 1 h, and a DAKO REAL EnVision Detection kit (DAKO, Glostrup, Denmark) was then used as the secondary antibody. After incubation in 3'3-diaminobenzidine for 5 min, the sections were counterstained with Mayer's hematoxylin and mounted. Negative controls were prepared by replacing the primary antibody with non-immune serum of the same species. They were assigned a score number according to the following rules: a score of 0 for 0% epithelium cells positive, a score of 1 for 1-24% epithelium cells positive, a score of 2 for 25-49% epithelium cells positive, a score of 3 for 50-74% epithelium cells positive and score of 4 for 75-100% epithelium cells positive. The intensity of cellular staining was also assigned a score number: a score of 0 for zero intensity, a score of 1 for weak intensity, a score of 2 for moderate intensity and a score of 3 for strong intensity(17). A staining score was obtained by multiplying the percentage score with the intensity score, with a maximum score of 12. Statistical evaluations were performed using the paired t-test. A difference considered statistically significance is 0.05.  Table 2.

Expressions of CRT and AnxA2 determined by 2-DE and 1-DE western blotting analysis
2-DE western blotting analysis of CRT and AnxA2 of cholesteatoma and retroauricular skin tissues of the patients was performed, and representative results are shown in Fig. 3. The results demonstrated that CRT and AnxA2 had higher expression levels in retroauricular skin, which was in agreement with the results presented in Fig. 1 and Fig. 2. Next, we compared the expression levels of CRT and AnxA2 in cholesteatoma and retroauricular skin tissues of five patients by 1-DE western blotting. Side-by-side comparison of the expressions of CRT and AnxA2 for each individual patient showed that the expressions of both proteins were obviously higher in retroauricular skin than in cholesteatoma ( Fig. 4), which was in accordance with the results of proteomic analysis.

Immunohistochemical distribution of CRT in cholesteatoma and retroauricular skin
CRT immunoreactivity in retroauricular skin showed a greater presence and a higher intensity than that in cholesteatoma tissue (Fig. 5). CRT immunoreactivity in retroauricular skin was clearly stronger than that in cholesteatoma. The results supported the findings of 1-DE and 2-DE western blot analysis.

Discussion
In this study, we found that CRT and AnxA2 were downregulated in cholesteatoma tissue as compared with their expressions in retroauricular skin tissue of the same patient. CRT is a 46-kDa protein with high-capacity Ca 2+ -binding and a multifunctional protein that has been shown to regulate several important cellular processes. It is mainly located in the endoplasmic reticulum and binds to newly- Overexpression of CRT in tumors in relation to normal tissue has been reported in human breast carcinoma(23-26), pancreatic cancer(27), prostate cancer(28), bladder cancer(29), hepatocellular carcinoma(30), gastric cancer (31), and oral cancer (32). CRT can also promote cell proliferation, cell differentiation and invasion in acute myeloid leukemia cells (33), and cell proliferation and migration in Schwan cells (34).
Previous research revealed that GRP75, GRP78 and GRP94 were upregulated in cholesteatoma, which may act against stress in the endoplasmic reticulum, avoiding cell apoptosis and protein unfolding and leading to increased growth of cholesteatoma(17). CRT is also an endoplasmic reticulum chaperone and is involved in endoplasmic reticulum stress to prevent protein unfolding, but is downregulated in cholesteatoma. Fischer et al. (35) reported that CRT inhibited lipopolysaccharideinduced inflammatory osteoclastogenesis and bone resorption. With the lytic enzymes and cytokines produced in cholesteatoma, the inflammatory condition may further promote epithelial proliferation in cholesteatoma. We speculated that the reduced expression of CRT and the inability to inhibit the persistent inflammatory response induces epithelial proliferation in cholesteatoma.
AnxA2 is a 36-kDa calcium-dependent phospholipid-binding protein. It is involved in several biological processes, such as immune responses, anti-inflammatory effects, Ca 2+ transport, Ca 2+ -dependent exocytosis, and phospholipase A2 regulation (36). It also plays roles in the regulation of cellular growth, cell division and signal transduction pathways (37,38). AnxA2 has been shown to interfere with multiple cellular processes, and particularly in cancer progression. AnxA2 is overexpressed in numerous types of cancer, including colorectal cancer, hepatocellular cancer, esophageal cancer, oral cancer, gastric cancer and pancreatic cancer (39)(40)(41)(42)(43)(44). It is also a serum marker for hepatocellular carcinoma, and has been suggested to have an important role in liver cancer progression.
Downregulation of AnxA2 in hepatocellular carcinoma cells has been shown to inhibit cell migration and cell invasive potential, and interfere with cytoskeleton establishment in tumor cells (40). It has been reported to be a useful biomarker for oral cancer (39). Previous study demonstrated AnxA2 participation in cell proliferation in orbital fat tissue of thyroid orbitopathy patients (45).
Comprehensive research showed that AnxA2 plays an important role in cell proliferation. Kim et al.

Conclusions
AnxA1 is the first protein family member of the annexin family that binds to the cell membrane in a calcium-dependent manner. ANXA1 may be induced by glucocorticoids in inflammatory cells and share many anti-inflammatory effects with these drugs(47). AnxA2 also have anti-inflammatory effects. It is speculated that the reduced expression of AnxA2 and inhibited the inflammatory 46.