Studies On The Role Of PRMT5in Biological Behaviors Of Epithelial Ovarian Cancer

Ovarian cancer is one of the leading causes of death from gynecological malignancies. Over80%of ovarian cancers are of epithelial origin (EOC), which show highly invasive, respond poorly to chemotherapies and are usually detected at advanced stages, resulting in poor prognosis. Despite the improvement of surgical techniques and the advent of more effective therapeutics, the overall5-year survival of epithelial ovarian cancer has increased only from36%to44%during the last three decades. To improve better prognostic and predictive assays and develop new therapeutic strategies for HOC, understanding the molecular mechanisms underlying HOC is critical.Arginine methylation is becoming an important type of posttranslational modification and is widely appreciated, playing a vital role in the regulation of diverse cellular procession that range from transcription and RNA procession to signaling, differentiation, apoptosis and tumorigenesis. The methylation of arginine residues is catalyzed by the protein arginine methyltransferase(PRMTs) family of enzymes. To date, PRMTs have been found11members in human, most of PRMTs except for PRMT2, PRMT10, and PRMT11have been shown to have enzymatic activity and can catalyze arginine methylation. Based on the type of modification they catalyze, PRMTs are mainly classified as type1and type11enzymes. All these two enzymes catalyze the formation of a mono-methylated (MMA) intermediate, subsequently, type I PRMTs (PRMT1,3,4,6and8) further catalyze the production of asymmetric dimethylation of arginine residues (aDMA), and type II PRMTs (PRMT5,7and PRMT9) catalyze the formation of symmetric dimethylation of arginine residues (sDMA).Protein arginine methyltransferase5(PRMT5) the first identified type II enzyme, is in human chromosome14q11.2and has two isoforms with637and620amino acids in length. It is involved in a variety of biological processes including ribosome biogenesis, assembly of the Golgi apparatus, cellular differentiation, germ cell specification, cellular proliferation, and apoptosis. In addition, it has been shown to interact with many other genes, such as cyclin E1, ST7and NM23, p53, E2F-1, PDCD4and E-cadherin to participate many cell processes. In human, aberrant expression of PRMT5are found in a wide variety of human diseases, especially in many cancers, including leukemia and lymphoma, breast cancer, colorectal cancer and lung cancer. Moreover, it is relevant to poor outcome in breast cancer and colorectal cancer patients.However, little is known about the role of PRMT5in ovarian cancer. In this study, we investigated the role of PRMT5in epithelial ovarian cancer.Part I:PRMT5is Overexpression in Epithelial Ovarian Cancers and is Associated with Poor Disease PrognosisObjective:1. To detect the difference of PRMT5expression in normal epithelium cells, epithelial ovarian benign tumors, borderline tumors and ovarian cancers.2. To valuate the relationship between PRMT5overexpression and clinicopathologic features of the patients.3. To examine the same set of EOC specimens with Ki-67, and investigate the relationship between PRMT5and Ki-67.4. Evaluate the relevance of PRMT5expression to the prognosis of ovarian cancer patients. Methods:1. EOCs (n=118), epithelial ovarian benign tumors (n=20), borderline tumors (n=14) and normal ovaries (n=12) samples were collected from the Department of Gynecology, Qilu Hospital of Shandong University between January2005and December2008. The clinical and follow-up data of ovarian cancer patients wre collected. The study was approved by the Institutional Medical Ethics Committee of Qilu Hospital of Shandong University and informed consent were obtained by patients.2. PRMT5and Ki-67expression were examined by immunohistochemistry (IHC) of standard streptavidin-peroxidase complex method.3. All statistical analyses were performed with SPSS13.0statistical software. IHC data were analyzed using Chi-square test or Fisher’s exact test. The Spearman test was used to analyze the relationship between PRMT5and Ki-67scores. Univariate and multivariate Cox regression analyses were used to evaluate the prognostic significance of PRMT5and other variables of ROC. Kaplan-Meier method was used to calculate the survival curves, and Log-rank test was used to compare the overall survival and progression-free survival between patient subgroups.Results:1. The positive staining of PRMT5was observed both in nuclear and cytoplasm of cells. High expression of PRMT5was observed in83.1%(98/118) of EOCs, in33.3%(4/12)of normal ovaries, in30%(6/20) of benign tumors, in64.3%(9/14) of borderline tumors. The level of PRMT5in EOCs was higher than normal ovaries and benign tumors (P<0.05, respectively), and there was no significant difference between borderline tumors and ovarian cancers.2. High expression of PRMT5was significantly associated with serous subtype, advanced FIGO stage(Ⅲ/Ⅳ), poor differentiation(G3), lymph node invasion, and presence of residual tumor (P<0.05, respectively).3. There was a significant positive correlation between PRMT5and Ki-67expression (r=0.377, P<0.001) in ovarian cancers, in which tumors with high Ki-67expression had a significantly higher expression of PRMT5(69.2%vs.98.2%, P<0.001).4. At the end of the follow-up period, of the118ovarian cancer patients, the median OS time was40months (95%CI:19.1-60.1), and median PFS time was20months (95%CI:16.8-23.2).5. In Univariate Cox analyses, serous subtype, high PRMT5expression, older age (>60years), advanced FIGO stage (Ⅲ/Ⅳ), and poor tumor differentiation (G3) were unfavorable predictors of OS (P<0.05, respectively), and all clinico-pathological variables, except lymph node invasion, were unfavorable predictors of PFS (P<0.05, respectively). Kaplan-Meier survival curves indicated that patients with high PRMT5expression had a significantly poor OS and PFS than those with low PRMT5expression (Log-rank test, P<0.001, respectively).6. In multivariate Cox analysis, the high expression of PRMT5, older age and advanced FIGO stage retained their significance as independent prognostic factors of poorer OS and the high expression of PRMT5, advanced FIGO stage and presence of residual tumor were found to be independent prognostic factors for shorter PFS.Conclusions:1. The expression of PRMT5was increased gradually between ovarian benign, borderline and malignant tumors and overexpression of PRMT5was associated with poor clinicopathologic features of the patients. These results demonstrate that PRMT5may play a crucial role as an oncogene in the development of EOC.2. The expression of PRMT5was positively correlated with the expression of Ki-67, which indicated that PRMT5may be involved in the regulation of ovarian cancer growth and proliferation.3. In univariate and multivariate survival analyses, high level of PRMT5was identified as an independent factor for poor prognosis in patients with EOC. Part II:Effects of PRMT5-silencing on Proliferation, Apoptosis, Migration and Invasion of Ovarian Cancer CellsObjective:To investigate the effects of PRMT5-siRNA-mediated gene silencing on proliferation, apoptosis, migration and invasion of ovarian cancer cell lines A2780and SKOV3. To explore the role of PRMT5in ovarian cancer cells in vitro, and to develop therapeutic potential of PRMT5in ovarian cancer.Methods:1. The two human PRMT5siRNA sequences siP1and siP2, control-siRNA(siC) were designed and chemically synthesize. They were transfected into ovarian cancer cell lines A2780and SKOV3with LipofectamineTM2000.2. Silencing of PRMT5was assayed for mRNA and protein expression level at48hr and72hr after transfection, respectively. The most efficient and specific sequence to silence the expression of PRMT5in two cells was screened.3. CCK8, BrdU incorporation and Ki-67labelling assays were used to detect the proliferation activity of ovarian cancer cells after PRMT5-siRNA was transfection.4. Annexin V-FITC/PI staining by How cytometry analysis was used to detect the apoptosis of ovarian cancer cells after PRM15-siRNA was transfection.5. Transwell assay was employed to test ovarian cancer cell migration and invasion after PRMT5-siRNA was transfection.6. E2F-1,eIF4E and E-cadherin protein were detected by Western blot assay after PRMT5-siRNA was transfection.7. Quantitative data were expressed as means±SD, the significance of the difference between groups was evaluated with independent-samples t test. All calculations were performed using SPSS13.0statistical software.Results:1. Two PRMT5siRNA (siP1and siP2) and scrambled control (siC) were respectively and successfully transfected into ovarian cancer cell line, and the efficiency of transfection was up to90%.2. Compared with normal control or cells transfected with siC, siP1could reduce PRMT5mRNA and protein in A2780and SKOV3cells significantly; siP2could reduce PRMT5expression in SKOV3, but the silence effect of siP2in A2780was weak. According to the results, siPl was the most efficient and specific sequence to silence the expression of PRMT5in two cell lines.3.72hr after transfection, the cell viability started to show lower in the cells transfected with siPl as compared with the cells transfected with siC and normal control (P<0.05). Similar results were observed in both A2780and SKOV3cells lines. The inhibition of growth was in a time-dependent manner.120hr after transfection, the cell viability of siPl group was24.4±0.9%in A2780, and was36.9±1.7%in SKOV3, both of them were significantly lower than the cell viability in siC group (P<0.001, respectively)4.48hr after transfection, compared to control sample that transfected with siC, cells treated with siPl showed significantly decreased BrdU incorporation,11.7±1.5%vs.33.3±1.5%in A2780, and18.0±2.0%vs.35.3±1.5%in SKOV3(P<0.05, respectively).5.72hr after transfection, compared to siC, cells transfected with siPl showed significantly decreased positive percentage of Ki-67,12.4±3.3%vs.50.2±8.0%in A2780,2.3±1.0%vs.23.6±6.6%in SKOV3(P<0.001, respectively).6.72hr after transfection, the percentage of apoptosis cells in siPl cells was higher than that in siC cells (29.4±0.9%vs.16.3±0.8%, P<0.001) in A2780; the percentage of apoptosis was28.9±0.6%vs.14.1±1.4%(P<0.001) in SKOV3.7. Both tumor cell migration and invasion assays indicated that transfection of PRMT5-siRNA could reduce the migration and invasion power of ovarian cancer cells. The number of migration/invasion cells with siPl group was lower than that with siC and normal control (P<0.001, respectively).8. E2F-1and E-cadherin protein were increased after PRMT5-siRNA transfection in ovarian cancer cells.Conclusions: 1. PRMT5could significantly promote the proliferation, migration and invasion, inhibit the apoptosis of ovarian cancer cell line, suggesting that PRMT5could play an important role in the tumorigenesis and development of ovarian cancer. PRMT5may be a novel therapeutic target for epithelial ovarian cancer.2. PRMT-siRNA could inhibit the proliferation, induce apoptosis via up-regulation E2F-1and reduce the migration/invasion ability via up-regulation E-cadherin in ovarian cancer.