The Expression And Functional Mechanism Of HrtA2/Omi In Epithelial Ovarian Cancer

Part one: the expression and clinical value of Hrt A2/Omi in epithelial ovarian cancerPurpose: Detection of Htr A2/Omi levels both in epithelial ovarian cancer tissue and cell lines and exploration its relationship with clinical pathological features.Methods: 1.Detection the expression of Htr A2/Omi in 60 cases of normal ovarian epithelial tissue and 156 cases of epithelial ovarian cancer tissue using immunohistochemical staining.2.Detection the expression of Htr A2/Omi in 60 cases of normal ovarian epithelial tissue and 156 cases of epithelial ovarian cancer tissue using real-time PCR.3.Analysis of the relationship between the expression of Htr A2/Omi and clinical pathological features in epithelial ovarian cancer patients.4.Detection the expression of Htr A2/Omi both in epithelial ovarian cancer tissue and cell lines using real-time PCR and immunohistochemical staining respectively.Results: 1.The expression of Htr A2/Omi in epithelial ovarian cancer tissue was significantly higher than normal ovarian epithelial tissue(P<0.001).2.The m RNA expression of Htr A2/Omi in epithelial ovarian cancer tissue was significantly higher than normal ovarian epithelial tissue(P<0.001).3.The expression of Htr A2/Omi was closely related with FIGO staging,lymph node metastasis,and had no obvious correlation with patient's age and tumor differentiation degree(P<0.001).4.The m RNA and protein expression of Htr A2/Omi in ovarian cancer cell lines OVCAR-3 and A2780 were significantly higher than normal ovarian epithelial cells(P<0.001).Conclusions: 1.The expression of Htr A2/Omi in epithelial ovarian cancer tissue was higher than normal ovarian epithelial tissue.2.The expression of Htr A2/Omi was closely related with FIGO staging,lymph nodemetastasis,and had no obvious correlation with patient's age and tumor differentiation degree.3.Htr A2/Omi was high expressing in both A2780 and OVCAR-3 cell lines.Part two: The experimental research of the influence of Htr A2/Omi gene silencing to apoptosis,invasion and metastasis in OVCAR-3-the ovarian cancer cellsPurpose: We observed its interference in cell apoptosis and invasive ability after silencing the expression of Htr A2/Omi in ovarian cancer cell lines-OVCAR-3 using RNA interference.Methods: 1.Detection the expression of Htr A2/Omi using real-time PCR and Western blot after transfecting OVCAR-3 cells.2.We observed the change of cell apoptosis using MTT method after Htr A2/Omi gene silencing.3.We observed the cell invasive ability using transwell invasive chamber after Htr A2/Omi gene silencing.Results: 1.Compared with si RNA-CN group and control group,the expression of Htr A2/Omi was significantly decreased in OVCAR-3/si RNA-Htr A2/Omi group in m RNA and protein level respectively(P<0.01).2.Compared with si RNA-CN group and control group,the apoptosis rate was significantly decreased starting from the third day in OVCAR-3/si RNA-Htr A2/Omi group(P<0.05).3.Compared with si RNA-CN group and control group,the number of cells passed artificial basement membrane were significantly decreased in OVCAR-3/si RNA-Htr A2/Omi group(P<0.01).Conclusions: 1.Gene silencing of Htr A2/Omi could decrease the apoptosis rates of OVCAR-3 cell lines.2.Gene silencing of Htr A2/Omi could increase the invasive ability of OVCAR-3 cell lines.Part three: the influence of Htr A2/Omi expression on growth of OVCAR-3 nude mouse transplantation tumorPurpose: We constructed subcutaneous transplantation tumor model by inoculated OVCAR-3/si RNA-Htr A2/Omi and si RNA-CN cells in nude mice respectively to observe tumor growth,cell apoptosis and the expression of apoptosis proteins.Methods: 1.Established subcutaneous transplantation tumor model: choosing healthy purebred nude female BALB/C mice(10-20 g,age 4-5 weeks),feeding in a germ-free SPF animal feeding equipment.And 60 nude mice were divided into 2 groups randomly,the experimental group was subcutaneously inoculated OVCAR-3/si RNA-Htr A2/Omi cells and the control group was subcutaneously inoculated si RNA-CN cells.2.Observed the general condition of nude mice daily and measured both of long and short diameters of tumor nodules weekly and calculated the volume of tumor after established the model.All nude mice were killed to weight the tumor after continuous three weeks of feeding.3.Detection of cell apoptosis rates in tumor tissue using TUNEL method.4.Detection the expression of apoptosis protein in subcutaneous transplantation tumor model using real-time PCR and Western blot respectively.Results: 1.Experimental nude mice began to appear palpable tumors and tumor grow rapidly after 1 week inoculation,slow growth in nude mice 3 weeks later and nude mice began to reduce weight and activity.While,control nude mice began to appear palpable tumors and tumor grow slowly after 3 week inoculation,and nude mice had a good appetite,weight growth faster and more activities.All nude mice were survived and administered successful after 3 weeks inoculation.2.All nude mice were killed and removed subcutaneous transplantation tumor to measuring the length and weight of tumor.The tumor volumes were 1673±105.6mm3 and 578±183.4mm3 respectively in experimental group and control group,and the tumor volume was significantly greater than control group(P<0.001).The tumor weights were 2.48±0.24 g and 0.91±0.25 g respectively in experimental group and control group,a nd the tumor volume was significantly greater than control group(P<0.001).3.TUNEL results show that the number of brown nuclear cells in experimental group greater than control group and the distribution densely.Compared with control group(34.63±1.91%),the AI in experimental group was 6.04±0.19% and had statistically significant difference(P<0.01).4.Compared with control group,the expression of Htr A2/Omi protein,XIAP protein,caspase-3 fragment and caspase-9 fragment were decreased in experimental group.Conclusions: 1.Down-regulation of Htr A2/Omi could promote tumor growth of OVCAR-3 nude mice subcutaneous transplantation tumor.2.Down-regulation of Htr A2/Omi could decrease cell apoptosis of OVCAR-3 nude mice subcutaneous transplantation tumor.3.Down-regulation of Htr A2/Omi could inhibit ovarian cell apoptosis by decrease the expression of XIAP,caspase-3 and caspase-9.