Differential Proteomic Analysis Revealed Cyclophilin A As A Candidate Of Human Endometrial Carcinoma-associated Protein

Objective: Protoemics has been one of the most important approaches in the researches of cancer pathogenesis, biomarker screening, target to pharmacological action and gene interference, characterized by the analysis of global protein changes in certain tissue or cell in integrity and high flux. The present study was proposed to separate the global protein in endometrial carcinoma and normal endometrium tissues by 2-DE and analyze the proteins with differential expression in 2-D profiles, and identify them with MS. The selected carcinoma-associated candidate was then validated by RT-PCR, Western Blot, and IHC assays。Its activity in cell proliferation and apoptosis was valued with RNA interference. We aimed to propose some novel projects with fine prospects to the insight of endometrial carcinoma pathogenesis, screening of biomarkers and gene intervention targets.Methods: The global proteins of eight endometrial carcinoma tissue specimens and their coupled normal endometrium were extracted and separated by 2-DE. The 2-DE image profiles were analyzed and valued with PDQuest 7. 1. 1 software, including cut, spot detection and match analysis. Then those spots with differential expressions were cut for in-gel digestion, The peptides were identified by MALDI-Q-TOF MS/MS and data were searched against protein database. Those proteins, with stable expression, constant and significant difference and perfect MS result, were selected as candidates to endometrial carcinoma-associated protein. RT-PCR and Western Blot were performed to validate the differential expression of the candidate between carcinoma and normal tissues. Furthermore, the differences were confirmed by IHC with paraffin specimens of 52 endometrial carcinoma, 39 normal endometrium, 16 cervical carcinoma and 10 normal cervix tissues. The results were analyzed statistically with semi-quantitive scores and IOD by Imagepro-Plus5.0 software. The siRNA targeted to the candidate was then transfected by Lipofectamine2000 to the endometrial carcinoma HEC-1-B cells to inhibit the gene RNA expression. The cell proliferation, apoptosis and cytocycle distribution were measured by MTT, clonogenic formation test, and flow cytometry to evaluate its biological activities.Results: Eight couples of 2-D patterns with high resolution and reproducibility were obtained, 782±28 and 760±33 spots detected in carcinoma and normal tissues, respectively, with intragroup match ratio (%) of 85.625±4.2. 112 differential spots were selected, 60 up-regulated and 52 down-regulated in carcinoma, and 99 of them (88.4%) were identified by MS. By analysis and classification of those proteins, cyclophilin A was considered to be the endometrial carcinoma-associated protein candidate. RT-PCR showed that expression of cyclophilin A and its receptor CD 147 in carcinoma were 2.49- (P＜0.01) and 3.69-fold (P＜0.01) higher than in normal tissues, and Western blot also showed 2.55-fold higher expression in carcinoma than in normal tissues (P＜0.01), which validated the reliability of the identified results. IHC showed cyclophilin A expression in endometrial carcinoma as: negative 0, weakly positive 9.6%, positive 44.2% and intensive positive 46.2%; normal endometrium as: negative 23%, weakly positive 77%, positive and intensive positive 0 (P＜0.01). IOD of normal and carcinoma tissues were 5.991±1.515 and 34.028±6.91 in average (P＜0.01). Relevance was suggested to the pathological classification (P＜0.01), but not to surgical-pathological staging (P＞0.05). Similar outcomes were observed in cervical carcinoma and normal cervix tissues. Transfection of cyclophilin A-siRNA into endometrial carcinoma HEC-1-B cells resulted significant inhibition of cyclophilin A expression with inhibition ratio over 60%. The proliferative activity of transfected ceils decreased significantly in dose- and duration-dependent manners, with inhibition ratio reaching 52.4% at 100nM after 72 hours (P＜0.01). The clonogenic formation test also showed the lower cloning number and cloning efficiency in transfected cells after 14-day culture, in comparison with cells in control (P＜0.01). Apoptosis was also induced by the siRNA transfection, with apoptotic ratio (%) reaching 45.7±3.1 after 72 hours (P＜0.01), accompanied with G₀/G₁ stage arrest and S stage decrease in cytocycle.Conclusion: The present study obtained the perfect 2-D protein profiles of endometrial carcinoma and normal endometrium tissues. 99 of the 112 selected spots with differential expression were identified by MS analysis and cyclophilin A was determined to be the endometrial carcinoma-associated protein candidate. The significant differences in expression between carcinoma and normal tissues were verified and confirmed by RT-PCR, Western blot and IHC. In addition, the relevance to the pathological classification, but not to surgical-pathological staging was also observed. Inhibition of cyclophilin A expression by RNA interference suggested its potential activities involved in cell proliferation and apoptosis process. These results might introduce some novel ideas and projects to the research of endometrial carcinoma pathogenesis, and identify cyclophilin A as potential biomarker and gene intervention targets of endometrial carcinoma.