Studies On The Expression Of Pim-3 In Ovarian Cancer And The Effects Of Pim-3 Gene Over-Expression On The Ovarian Carcinoma Cell SKOV3

In recent years,studies found that Pim-3 can inhibit apoptosis,promote cell proliferation and affect the cell cycle and play an important role in the occurrence and development of tumor.Pim-3 has been a new hot spot in tumor research and in-depth study of its function will bring a revolutionary change to understand the regulation network of tumor development.Pim-3 is abnormally expressed in liver cancer,colon cancer,gastric cancer and other tumors and is related with proto-oncogene and postoperative recurrence.Pim-1 is highly expressed in epithelial ovarian cancer and plays a role of antiapoptotic,but that Pim-3 expression in ovarian cancer and its role in ovarian cancer occurrence and development are still a subject to be further studied.Object:1.To explore the Pim-3 expression and their relationship with the ovarian cancer clinical pathologic factors;2.Construct plasmid pCDH-Pim-3 and transfect it into the SKOV3 cells to established the Pim-3 overexpressed stable cell line.3.To explore the function of Pim-3 involved in cell proliferation,migration and apoptosis in SKOV3 cells.Methods:1.Using Realtime RT-PCR method,Pim-3 gene expression were detected in 26 cases of ovarian cancer,25 cases of ovarian benign tumors and 16 cases of normal ovarian tissue specimens,respectively;The different expression levels of Pim-3 gene among the three groups and their relationships with the clinical pathological characteristics of ovarian cancer were analyzed by ANOVA with SPSS 17.0 software.2.Pim-3 cDNA was amplified by RT-PCR and the amplification products were cloned into pCDH-Pim-3 plasmid to construct eukaryotic fluorescent expression vector.The constructed plasmid was transfected into SKOV3 cells to establish the Pim-3 overexpressed monoclonal cell line.The expression level of Pim-3 was determined by realtime RT-PCR at mRNA levels and Western Blot at protein levels.3.The impacts of Pim-3 expressed on cell proliferation,apoptosis and migration in SKOV3 cells were detected by CCK8,flow cytometry and scratch test,respectively.Results:1.The expression of Pim-3 in ovarian cancer tissue(2.88 ±4.57)was significantly higher than that of benign ovarian tumor(0.065 ± 0.04)and normal ovarian group(0.028 ±0.015)and differences were statistically significant among the three groups.The expression of Pim-3 gene in ovarian cancer tissue is statistically unsignificant with age(P = 0.53),the presence of abdominal cavity effusion(P =0.07),and were statistically significant with the clinical stage(P = 0.02),patholog:ical grade(P = 0.03),and presence of metastasis(P = 0.03)(P<0.05),respectively.2.pCDH-Pim-3 plasmid was successfully constructed.Green fluorescence was observed under fluorescence microscopy after transfection of SKOV3 cells.Realtime RT-PCR and Western Blot results showed that expression level of Pim-3 was higher than that of pCDH vector group(P<0.05).3.The results of CCK8 test showed that OD450 values of pCDH-Pim-3 plasmid transfected cells were 0.53±0.07,2.17±0.03 and 3.67±0.25 after 24hours?48 hours and 72 hours,respectively,wheares those of pCDH vector group were 0.44±0.03,1.53±0.03 and 2.64±0.40,respectively.However,migration of SKOV3 cells were 4.00±0.33mm after transfection of Pim-3 constructed plasmid,while those of mock group and pCDH vector group cells were 3.21±0.23mm and 3.14±0.17mm(P<0.05).The apoptosis of SKOV3 cells was inhibited after after transfection of Pim-3 constructed plasmid.Conclusion:1.Pim-3 mRNA expression in ovarian cancer group is higher than those of benign ovarian tumor and normal ovarian group,the differences were statistically significant.And there was no statistical difference between benign tumor group and normal group.The expression levles of Pim-3 gene in ovarian cancer tissue has relationship with clinical stage,pathological classification,and presence of metastasis,but has no relationship with age and abdominal effusion.2.The constructed plasmid pCDH-Pim-3 was constructed and transfected successfully into the SKOV3 cells to establish the Pim-3 overexpressed stable cell line.3.The cell proliferation was upregulated and the cell migration were increased,the cell apoptosis was inhibited.Our results would be helpful to clarify the roles of Pim-3 in SKOV3 tumorigenesis and metastasis and would provide experimental data for finding new clinical diagnosis and therapy targets of ovarian cancer.