| BackgroundOvarian cancer is one of the most common and possesses the highest mortality in gynecological cancers. In all the histological types, epithelial carcinoma is the most common, accounting for above90%. Most of ovarian epithelial carcinoma patients are found to be advanced stage at the first diagnosis due to a lack of early symptoms. Although the progression on surgery and chemotherapy, most of patients with complete remission become recurrence in about two years. All cases of recurrent ovarian cancer are nearly drug-resistant and incurable. To search for biomarkers associated with epithelial ovarian carcinoma is of importance for early diagnosis and overturn of drug-resistance of the disease.Since the first tumor biomarker was found in1948, the progression has been very quick. But both sensitivity and specificity of all biomarkers, including CA125, CA199, CA153and HE4, are limited for ovarian epithelial carcinoma. A new scientific technology, Proteomics, appeared in the20th century. Proteomic analysis has been widely used in medical research. A lot of diseases including cancer have been regarded as abnormal proteomic diseases. Proteomics provides a useful tool for searching for biomarkers in cancer.Recently, a specific population, cancer stem cells (CSCs), in cancer tissues has been identified. CSCs were initially found in acute myloid leukemia in70’s of the20th century. Now, cancer stem cells have been identified in many solid tumors including ovarian cancer. Cancer stem cells possess the capacity of tumorigenesis, self renewal and differentiation, play a key role in the initiation and development of cancer, and are believed to be the resource of cancer metastasis and recurrence. Of importance, all cancer stem cells are resistant to chemotherapeutic drugs. To investigate the characteristics of protein expression profiles of cancer stem cells is of significance for cancer therapy targeting cancer stem cells.In a previous study of ours, we have isolated, enriched and identified ovarian cancer stem cell-like cells (OCSC-LCs) from ovarian cancer cell line3AO by serum-free culture, and found that those cells expressed stem cell markers, Notch2and Sox2, and presented CD24negative and drug-resistant. Previous study promotes our subsequent researches.Here, we initially screened and identified differentiated protein expressions in ovarian cancer stem cell-like cells and taxol-resistant cells, compared with their parental cells, using proteomic analysis and bioinformatics; then verified four differentiated expressed proteins in cell models by Western blot; and finally detected the expression of UTP23protein in ovarian epithelial carcinoma tissues by immunohistohemistry and analyzed the association of UTP23expression with clinicopathologic parameters and prognosis of patients. The aim of our study is to find potential biomarkers associated with ovarian cancer, and provide possible novel approach to the diagnosis and treatment for ovarian cancer. PartⅠ Isolation and identification of differentiated protein expression in ovarian cancer stem cell-like cells and taxol-resistant cellsObjective:To isolate and identify differentiated protein expression in ovarian cancer stem cell-like cells and taxol-resistant cells compared with their parental cells, and search for potential biomarkers associated with ovarian cancer.Methods:Ovarian cancer stem cell-like cells were obtained from3AO spheroid cultured in serum-free. Taxol-resistant cells (SKOV3-TR30) were obtained from SKOV3cultured with persistent and low concentration of taxol treatment. Differentiated protein expressions were assayed by Label free quantitative proteomic and two-dimensional differential in-gel electrophoresis, respectively. Function analysis was performed by Gene ontology and pathway was analyzed by KEGG method.Results:1. Label free quantitative proteomic(1) Between SKOV3-TR30and parental SKOV3cells, totally5015peptides and558proteins were identified.356statistically identified proteins, of those,47up-regulated and309down-regulated proteins.(2) Between ovarian cancer stem cell-like cells and parental3AO cells, totally3601peptides and431proteins were identified,187statistically identified proteins, of those,72up-regulated and115down-regulated proteins.2. Gene ontology analysis(1) In SKOV3-TR30and parental SKOV3cells, involved differentiated expressed proteins mainly included metabolic process (35%), regulation of biological process (24%) and Transport (13%)(2) In ovarian cancer stem cell-like cells and parental3AO cells, involved differentiated expressed proteins mainly included metabolic process (20%), regulation of biological process (12%) and cellular component organization (9%)3. KEGG ananlysisThere were no significant differences of pathways enrichment between SKOV3-TR30and parental SKOV3cells, and between ovarian cancer stem cell-like cells and parental3AO cells.4. Two-dimensional differential in-gel electrophoresis(DIGE) analysisThere was217differentiated expressed proteins between ovarian cancer stem cell-like cells and parental3AO cells, and225differentiated expressed proteins between SKOV3-TR30and parental SKOV3cells.Conclusions1. There are multiple differentiated expressed proteins between ovarian cancer stem cell-like cells and parental3AO cells, and between SKOV3-TR30and parental SKOV3cells.2. Involved differentiated expressed proteins mainly include metabolic process, regulation of biological process, transport, differentiation and others. Part Ⅱ Verification of differentiated expressed proteinsObjectives:To verify selected differentiated expressed proteins by proteomics, for providing the basic of subsequent research.Methods:The expressions of three down-regulated proteins (UTP23、FLNA、 HDGFRP3) and one up-regulated protein (ALDOC) were detected by Western blot in SKOV3/SKOV3-TR30,A2780/AT2780and3AO/3AO-SPH cells.Results:1. UTP23protein Compared with SKOV3, A2780and3A0, the expressions of UTP23protein were significantly decreased in SKOV3-TR30, AT2780and3AO-SPH P<0.01, p<0.001, p<0.05). The results were consistent with proteomics.2. FLNA protein Compared with SKOV3, A2780and3A0, the expressions of FLNA protein were significantly decreased in SKOV3-TR30,AT2780and3AO-SPH (P<0.005, P<0.001,P<0.05). The results were consistent with proteomics.3. HDGFRP3protein Compared with parental cells, part of the results were consistent with proteomics.4. ALDOC protein Compared with parental cells, all of the results were inconsistent with proteomics.Conclusions:1. The expressions of UTP23and FLNA protein are down-regulated in ovarian cancer stem cell-like cells and taxol-resistant cells, suggesting that both proteins may act as potential candidate biomarkers, and may be associated with maintaining the characteristics of ovarian cancer stem cells and modulating taxol-resistance.2. A further study is needed about inconsistence of the expressions of HDGFRP3and ALDOC protein between Western blot and proteomics. Part Ⅲ The expression of UTP23protein in ovarian epithelial carcinoma tissues and clinical significanceObjectives:To evaluate potential value of UTP23as biomarker by analyzing the expression of UTP23protein in ovarian epithelial carcinoma tissues and the association with clinicopathological parameters and prognosis of patients.Methods:The expression of UTP23protein was detected by immunohistochemistry in145ovarian epithelial carcinoma tissues, and the association of UTP23expression level with clinicopathological parameters and prognosis of patients was analyzed, including age, primary surgery, FIGO stage, tumor grade, ascites, presurgical CA125level,chemotherapy response, PFS and OS.Results:1. UTP23protein was expressed with brown staining in cytoplasm of ovarian epithelial carcinoma cells.2. Univariant analysis showed that low-expression of UTP23was significantly associated with higher FIGO stage, higher grade, suboptimal surgery, CA125>500U/L and drug-resistance (P<0.05).3. Univariant analysis showed that UTP23expression was significantly associated with OS (χ2=4.548, P=0.033) and PFS (χ2＝3.913P=0.048). Lower UTP23expression presented shorter survival. Conclusions:1. Ovarian epithelial carcinoma cells express UTP23with cytoplasm location.2. UTP23expression in drug-resistant is lower than that in drug-sensitive, suggesting that UTP23might be involved in drug-resistence of ovarian epitheial carcinoma.3. Lower expression of UTP23is associated with poorer prognosis, suggesting that UTP23could be used for predicting the prognosis of ovarian cancer pateints. |
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