| Epithelial ovarian cancer(EOC)displays very high morbidity and is the leading cause of death in patients with gynaecologic malignancies.Chemotherapy resistance and tumor relapse are two major causes of treatment failure of late-stage EOC.Meanwhile,several studies have established a connection between Cancer stem cell(CSC)and chemotherapy resistance and tumor relapse.Thus,it is necessary to study the resistance mechanism of CSC in EOC and find new prognostic biomarker for EOC diagnosis and theraptic target for clinical treatments.Acylglycerol kinase(AGK)is a novel lipid kinase,which is over expressed in a series of malignant carcinomas,such as cervical cancer,nasopharyngeal carcinoma,and breast cancers.Furthermore,our previous data showed that AGK was upregulated in EOC,and significantly correlated with the survival of patients.This study aims to investigate the biological function and molecular mechanism of AGK in the tumorigenesis and cancer stem cell proliferation of EOC cells.Objective1.Explore the expression of AGK in EOC;2.Analyze the relationship between AGK expression and the clinical pathological features of patients;3.Study the role of AGK in EOC cell proliferation;4.Study the role of AGK in EOC stem cell populations;5.Analyze the molecular mechanisms of AGK in EOC cell proliferation and EOC stem cell populations.Method1.To identify AGK expression,cDNA microarray analyzed 6 pairs of EOC patients and adjacent non-cancerous tissues(ANT);2.To investigate AGK expression in EOC,qRT-PCR and Western blotting analyzed one human normal ovarian epithelial cells and six human EOC cell lines and 4 pairs of EOC patients and ANTs;3.By IHC experiments,we further studyed the expression of AGK protein in 140 paraffin-embedded,archived EOC samples;4.We statistically analyzed the relationship between the expression of AGK in EOC specimens with clinical pathological features and survival of patients by SPSS 17.0;5.We established over-expression of AGK and AGK knocked down EOC cell models;6.In vitro,growth curve assay,colony formation assay and EdU experiments were used to demonstrate AGK can enhance EOC cellsproliferation;7.In vivo,a proliferation activity of AGK was further delivered with mouse xenograft model assay;8.qRT-PCR and Western-blotting were used to test the RNA and protein expression of AGK were closely related to the cell proliferation,such as p21,p27,cyclinD1 and Rb.9.We use qRT-PCR to test the expression of pluripotent associated markers.Sphere formation experiments were used to test the self-renewal capacity of cells.Flow cytometry experiments were used to test the SP+cells and analyzed the the stem cell population and stem cell-like phenotype in EOC.10.To identify AGK related gene expression,cDNA microarray analyzed 8 over-expression of AGK and AGK knocked down EOC cells;11.By AGK-BioID experiments,we analyzed the relationship between AGK and closely related protein.Result1.The result of cDNA microarray showed that AGK was overexpression in all EOC tissues.2.We found both AGK protein and mRNA expressions were markedly upregulated in all EOC cell lines,in comparison to that in one NOSC;The expression of AGK was significantly upregulated in all 4 EOC tissue samples(T)compared with that in paired ANTs obtained from the same patients;3.Further statistical analyses showed that AGK level was significantly associated with FIGO stage(P=0.002),the volume of ascites(P=0.001),peritoneal cytology(P=0.004);Moreover,we used Kaplan-Meier analysis and the log-rank test to perform survival analysis,and the survival time was significantly different between high and low AGK expression groups(P<0.05);4.Univariate and multivariate analyses showed that FIGO stage and AGK expression level was each recognized as an independent prognostic factor;5.Growth curveand colony formation assay experiments showed a drastic reduction of cell proliferation in OVCAR3 and CAOV3 cells in which AGK expression was overexpression or knocked down;6.Overexpression of AGK in OVCAR3 and CAOV3 cells enhanced the incorporation of EdU,but the silencing endogenous AGK reduced the incorporation of EdU.7.In vivo,OVCAR3/AGK tumors grew significantly faster than the OVCAR3/vector tumors throughout the experiment,whereas the tumors formed by OVCAR3/AGK-RNAi cells grew at a much slower rate than control OVCAR3/RNAi-Vector tumors;8.Western blotting analysis revealed that the expression levels of p21 and p27 were drastically reduced in AGK-overexpressing cells,which was accompanied by significantly increased expression of cyclinDl and phospho-Rb compared to control cells.Conversely,the expression of p21 and p27significantly increased whereas expression of cyclinDl and phospho-Rb significantly decreased in AGK-silenced cells.9.Overexpression of AGK enhanced the sphere formation ability,increased the SP+cells and promoted the expression of pluripotency associated markers in EOC cells.10.AGK expression level was associated with RPL39.Conclusion1.AGK was overexpression in all EOC tissues,and AGK expression level was recognized as an independent prognostic factor;2.AGK promoted the proliferation of EOC cells;3.AGK promoted the stem cell population and stem cell-like phenotype in EOC4.AGK expression level was associated with RPL39;5.AGK might be a novel and useful prognostic marker and a potential target for EOC treatment;6.AGK might be a novel EOC stem cell biomarker. |
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