| Epithelial cells play function through the cell polarity, adhesion and tight junction, at the same time limiting the ability of migration. Recently, researchers have found that epithelial cells can loose adhesion, tight junction and cell polarity in order to acquire the ability of invasive migration, defined as epithelial to mesenchymal transition (EMT). Accumulated evidences revealed shown that EMT were closely related with occurrence and development of malignant epithelial tumors. Recently, in the study of tumor biology, the two concepts of cancer stem cells and EMT are combined together. Our previous outcomes successfully isolated and identificated cancer stem-like cells (CSC-LCs) from epithelial ovarian cancer and preliminarily observed the EMT characteristics. Although the relationship between drug resistance of ovarian cancer and EMT has been reported, as well as the drug-Resistance of cancer stem cells, how the ovarian CSC-LCs undergo EMT process is still being undiscovered. Proto-oncogene Cyclin D1 plays a key role in maintaining undifferentiated state and anchorage-independent growth of the tumor cell, and several studies has found Cyclin D1 highly expression were closely related with metastasis and poor prognosis of diverse solid tumors, as we infer, which may play function in sustaining self-renewal and EMT ability of ovarian CSC-LCs. In this study, we used ovarian adenocarcinoma cell line 3AO as the research model, firstly proved the stem cell characteristics of CD24-cells from cell viability, cell apoptosis, cell migration and stem-related genes expression. Furthermore, we observed and studied the cell morphology,cell biology and possible function by Cyclin D1 effect of CD24-cells. This study was performed to elucidate the mesenchymal characteristics and EMT ability of ovarian CSC-LCs, which Cyclin D1 involved in, suggesting the EMT process of CSC-LCs participated in the invasion and metastasis of ovarian cancer.Part 1. The study on stem cell characteristics of CD24- human ovarian carcinoma cells in vitroObjective:To elucidate the stem cell characteristics of CD24- human ovarian carcinoma cells through comparision the cell viability, cell apoptosis, stem-related gene Bmi-1 and Oct-4 expression.Methods:The spheriods were induced by serum-free medium, and isolated by FACS into CD24- and CD24+ cells. MTT assay were used to observe cell viability, and PE-FITC-PI assay were carried out for apoptosis analysis. Stem-related genes Bmi-1 and Oct-4 were analysed by qRT-PCR.Results:1. A subpopulation of 3AO cell formed spheroids under serum-free medium with self-renewal ability;2. CD24- cells had stronger proliferative capacity than CD24+ under low serum conditions;3. CD24- cells presented stronger resistence of non-viable apoptosis induced by platinum;4. Stem-related gene Bmi-1 was highly expressed in CD24- cells and spheroids, gradually decreased during differentiation;5.Oct-4 expression was not different between CD24- and CD24+ cells.Conclusions:1. CD24- cells sustained proliferation in low serum environment, and tolerated induced apoptosis by platinum, which further proved the High proliferative capacity of the stem phenotype;2. Bmi-1 was highly expressed in CD24- and decreased during differentiation, which functioned with specificity for sustaining self-renewal characteristics in ovarian cancer stem-like cells;3. Oct-4 was expressed in CD24+, CD24-, and down-regulated during differentiation, which was not specific to ovarian CSC-LCs with the CD24- phenotype.Part 2. Cyclin D1 effect upon EMT ability of ovarian CSC-LCs in vitroObjective:To investigate the effect and possible mechanism of Cyclin D1 involved in sustaining EMT ability of ovarian CSC-LCs.Methods:The morphology of CD24- and CD24+ cells cultured in compelte medium were observed. Wound-repair assay was used to observe the migratory ability of CD24-enriched spheroids. The mRNA and protein levels of EMT-related moleculers E-cadherin and Vimentin were ananlysed by qRT-PCR studies. Using Cyclin D1-targeting siRNA to silence Cyclin D1 expression in 3A0 spheroids. Cell viability, cell apoptosis and wound-repair assay were carried out after transfection. The alteration of EMT related moleculers was ananlysed after transfection.Results:1. CD24- cells showed mesenchymal-shape, and transformed into madstone-shape during differentiation;2. CD24- enriched 3AO spheroids presented stronger migratory capability;3. CD24- cells seldom expressed E-cadherin but re-expressed during differentiation;4. CD24- cells highly expressed Vimentin and down-regulated during differentiation;5. Cyclin D1 transfected cells showed decreased proliferation ablility of 3AO spheroids;6. Cyclin D1 transfected cells showed decreased migration ablility of 3AO spheroids;7. Cyclin D1 transfected cells showed increased apoptosis ablility of 3AO spheroids;8. Cyclin D1 transfection resulted in reduction of EMT-related moleculers N-cadherin and Vimentin in 3AO spheroids.Conclusions:1. CD24- cells had characteristic of epithelial to mesenchymal transition (EMT), moreover, the EMT phenotype were gradually reversed during differentiation.2. Cyclin D1 may contribute to sustaining of self-renewal and EMT ability of 3AO CSC-LCs by proliferation promotion, migratory promotion and apoptosis inhibition.
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