Biological Behavior Of PDCD6and Its Effector Moleculars In Epithelia Ovarian Cancer

Ovarian cancer is the leading cause of death from gynecologic malignancy. Epithelial ovarian cancer accounts for90%of all ovarian cancer, the highest degree of malignancy. Due to lack of specific symptoms, insidious onset of ovarian cancer, progress rapidly, about75%of patients has emerged a wide range of intra-abdominal metastasis and ascites when they are first diagnosed. Most patients even remove the original lesion, but eventually died of recurrence and metastasis. In our previous study, two epithelia ovarian cancer cell lines with low and high metastatic potentials were set up successively, named as HO-8910, and HO-8910PM respectively. Seventeen different significantly expressed proteins between them were detected by comparative proteomic analysis, of which programmed cell death6(PDCD6) was identified. PDCD6, a calcium-binding protein belonging to the penta-EF-hand protein family, participates in T cell receptor-, Fas-, and glucocorticoid-induced programmed cell death. Recently, PDCD6was found up-regulated in several cancer tissue, especially in the metastatic cancer tissue, compared with normal tissue. Similarly, our results showed PDCD6was over-expressed in HO-8910PM which has higher metastatic potential, as compared with HO-89106. So, in order to further investigate biological behavior of PDCD6, association of its expression with clinical features and possible molecular mechanism of its biological effect in epithelia ovarian cancer, lentiviral vector with shRNA for PDCD6was used to knockdown the expression of PDCD6, and real time RT-PCR, western blot, CCK8, flow cytometry, Hoechst staining,transwell assay were employed to observe the effect of PDCD6on cell proliferation, cellcycle, apoptosis, drug sensitivity and cell motility. Our results showed compared with control, no obvious difference of cell morphology was found after HO-8910PM cells was infected72hours by Lentiviral shRNA-PDCD6. The growth rate of HO-8910PM was decreased. In the migration assay, compared with control, shRNA-PDCD6-HO-8910PM cells migrated to the bottom side of the membrane was markedly decreased (344±26vs.240±10), p<0.001. Same result was shown in invasion assay. Compared with control, cells invaded to the bottom side of the membrane was also significantly decreased (256±8vs.230±7), p=0.050. No evident effect of PDCD6on cell cycle, apoptosis and drug sensitivity was found. After that, real time RT-PCR was employed to detect mRNA expression of PDCD6in212ovarian cancer tissue samples. Combined with clinical and followup data, associations of PDCD6expression with clinical pathological factors, treatment response and survival were evaluated by rand sum test, Chi-square test, Kaplan-Meier survival curve and Cox regression statistical methods. No statistical significance of correlation between PDCD6mRNA expression and stage, grade, histology and treatment response in ovarian cancer was found, but the patients with stage III-IV or poor differentiated grade or serous tumor had higher PDCD6mRNA expression than those with stage Ⅰ-Ⅱ or well differentiated grade or non-serous tumor. PDCD6expression was positively correlate with residual tumor size (r=0.16, p=0.019). The proportion of high PDCD6expression in patients who had residual tumor after surgery was higher than that in patients who had no residual tumor,29.3%versus16.7%, p=0.050. No association was found between PDCD6expression and overall survival, but PDCD6mRNA expression was significantly associated with progression free survival. Patients with medium or high level of PDCD6mRNA had shorter progression free survival time than those with low levels. Kaplan-Meier survival analysis demonstrated5year survival rate of patients with low, medium or high level of PDCD6mRNA was56%,36%and41%and median survival time was43,38and21months respectively (x2=6.123, p=0.047). Multivariate Cox regression analysis showed PDCD6was an independent predictor for ovarian cancer relapse. Patients with medium or high levels of PDCD6mRNA were at higher risk for disease progression, compared to those with low levels (HR,1.29; P=0.024for mid levels; and HR,1.57; P=0.045for high levels) after adjusting for age, disease stage, tumor grade, histologic type and residual tumor size. Additionally, Illumina cDNA microarray was used to screen the interaction genes and signaling pathway with PDCD6. From the chip results, genes with very significant differences in expression and also closely related to tumor proliferation or metastasis were chosen to further confirm using real time RT-PCR. One hundred and thirty three differentially expressed genes reproducible were detected in2experiments. Among them,101genes were downregulated and32genes were upregulated with the silencing of PDCD6. Sixteen genes had very significant differences in expression (Diffscore＜-30or＞30, P＜0.001), such as ANGPTL4, IER3, CCND1, PDCD6, CNIH, BMP2, TGM2, SAA1, TRIM31, MYC, CD44, CXCL16, C3orf34, CAT, TNFRSF12A and METTL21A. Except for ENO2, the rest15genes were downregulated with the silencing of PDCD6. Two genes (CCND1, MYC) were associated with cell cycle and proliferation and7genes (ANGPTL4, TGM2, CD44, CXCL16, SAA1, TNFRSF12A and BMP2) involved in angiogenesis, cell migration and invasion. CCND1, MYC, ANGPTL4, BMP2and CXCL16were validated by real time RT-PCR, which might be effector moleculars of PDCD6. Furthermore, signaling pathway enrichment analysis showed classic MAP (ERK1/2) of MAPK signaling pathway were significantly enriched, the main activated genes as following:FGFR, MAP2K1/MEK1, MAP2K2/MEK2, MYC and FOS. Taken together, from the above studies on biological behavior of PDCD6, clinical association analysis and the differentially expressed effector molecules induced by PDCD6in epithelia ovarian cancer cells, we concluded that:1. PDCD6promoted cell proliferation and metastasis of epithelia ovarian cancer.2. Overexpression of PDCD6was correlated with malignant biological behavior of epithelia ovarian cancer.3. Patients with medium or high level of PDCD6mRNA had shorter progression free survival time and higher risk for disease progression than those with low level. PDCD6was an independent predictor for ovarian cancer relapse.4. PDCD6might upregulate CCND1, MYC, ANGPTL4, BMP2and CXCL16gene exprssion to promote ovarian cancer cell proliferation and metastasis through MAPK signaling pathway. PART Ⅰ The Effect of Downregulation of PDCD6Gene Expression in HO-8910PM on Cell Proliferation, Cell Cycle, Apoptosis, Drug Sensitivity, Migration and InvasionObjective:To investigate biological behavior of PDCD6in epithelia ovarian cancer.Methods:Lentiviral vector with shRNA for PDCD6was used to knockdown the expression of PDCD6. Real time RT-PCR, western blot, CCK8, flow cytometry, Hoechst staining,transwell assay were employed to observe the effect of PDCD6on cell proliferation,cellcycle, apoptosis, drug sensitivity and cell motility.Results:Compared with control, no obvious difference of cell morphology was found after HO-8910PM cells was infected72hours by Lentiviral shRNA-PDCD6. The growth rate of HO-8910PM was decreased. In the migration assay, compared with control, shRNA-PDCD6-HO-8910PM cells migrated to the bottom side of the membrane was markedly decreased (344±26vs.240±10), p<0.001. Same result was shown in invasion assay. Compared with control,cells invaded to the bottom side of the membrane was also significantly decreased (256±8vs.230±7), p=0.050. No evident effect of PDCD6on cell cycle, apoptosis and drug sensitivity was found.Conclusions:Downregulation of PDCD6in HO-8910PM resulted in slowing down cell proliferation and reducing cell migration and invasion. PART II Association Study of PDCD6Expression with Clinical Pathological Charateristics, Treatment Response and Prognosis in Epithelia Ovarian CancerObjective:To investigate relationship between PDCD6expression and clinicopathological factors, treatment response and prognosis in epithelia ovarian cancer.Methods:Two hundred and twelve fresh tumor tissue samples and relative clinical and followup data were collected. Real time RT-PCR was employed to detect mRNA expression of PDCD6in ovarian cancer tissue. Associations of PDCD6expression with clinical pathological factors, treatment response and survival were evaluated by rand sum test, Chi-square test, Kaplan-Meier survival curve and Cox regression statistical methods.Results:No statistical significance of correlation between PDCD6mRNA expression and stage, grade, histology and treatment response in ovarian cancer was found, but the patients with stage III-IV or poor differentiated grade or serous tumor had higher PDCD6mRNA expression than those with stage I-II or well differentiated grade or non-serous tumor. PDCD6expression was positively correlate with residual tumor size (r=0.16, p=0.019). The proportion of high PDCD6expression in patients who had residual tumor after surgery was higher than that in patients who had no residual tumor,29.3%versus16.7%, p=0.05. No association was found between PDCD6expression and overall survival, but PDCD6mRNA expression was significantly associated with progression free survival. Patients with medium or high level of PDCD6mRNA had shorter progression free survival time than those with low levels. Kaplan-Meier survival analysis demonstrated5year survival rate of patients with low, medium or high level of PDCD6mRNA was56%,36%and41%and median survival time was43,38and21months respectively(x2=6.123,p=0.047). Multivariate Cox regression analysis showed PDCD6was an independent predictor for ovarian cancer relapse. Patients with medium or high levels of PDCD6mRNA were at higher risk for disease progression, compared to those with low levels (HR,1.29; P=0.024for mid levels; and HR,1.57; P=0.045for high levels) after adjusting for age, disease stage, tumor grade, histologic type and residual tumor size.Conclusions:Overexpression of PDCD6was correlated with malignant biological behavior of epithelia ovarian cancer; Patients with medium or high level of PDCD6mRNA had shorter progression free survival time and higher risk for disease progression than those with low level; PDCD6was an independent predictor for ovarian cancer relapse. PART III Screening of the Interaction Genes and Signaling Pathway with PDCD6by cDNA MicroarrayObjective:Screening of the interaction genes and signaling pathway with PDCD6to investigate molecular mechanism of PDCD6in the proliferation and metastasis of ovarian cancer.Methods:Total RNAs were extracted from the experimental group cells (shRNA-PDCD6-HO-8910PM) and control cells (HO-8910PM-mock) after72hours infection by lentivirus. Biotin-labeled cRNA corresponding to the mRNA fraction was produced and then hybridized to the Illumina cDNA microarray.Illumina bead studio application software and DAVID database were used to do the biological informatics analysis. Experiment duplicated twice. From the chip results, genes with very significant differences in expression and also closely related to tumor proliferation or metastasis were chosen to further confirm using real time RT-PCR.Results:One hundred and thirty three differentially expressed genes reproducible were detected in2experiments. Among them,101genes were downregulated and32genes were upregulated with the silencing of PDCD6. Sixteen genes had very significant differences in expression (Diffscore<-30or>30,P<0.001), such as ANGPTL4, IER3, CCND1, PDCD6, CNIH, BMP2, TGM2, SAA1, TRIM31, MYC, CD44, CXCL16, C3orf34, CAT, TNFRSF12A and METTL21A. Except for ENO2, the rest15genes were downregulated with the silencing of PDCD6. Two genes(CCND1, MYC) were associated with cell cycle and proliferation and7genes (ANGPTL4, TGM2, CD44, CXCL16, SAA1, TNFRSF12A and BMP2) involved in angiogenesis, cell migration and invasion. CCND1, MYC, ANGPTL4, BMP2and CXCL16were validated by real time RT-PCR, which might be effector moleculars of PDCD6. Furthermore, signaling pathway enrichment analysis showed classic MAP (ERK1/2) of MAPK signaling pathway were significantly enriched, the main activated genes as following:FGFR, MAP2K1/MEK1, MAP2K2/MEK2, MYC and FOS. These results suggested PDCD6might relate with MAPK signaling pathway.Conclusions:PDCD6might upregulate CCND1, MYC, ANGPTL4, BMP2and CXCL16gene exprssion to promote ovarian cancer cell proliferation and metastasis through MAPK signaling pathway.