The Influence Of TCF21 On Proliferation And Apoptosis Of Ovarian Cancer Cells

The mortality of ovarian cancer tops the list in the gynecologic malignant tumors.As the etiology of ovarian cancer is hidden,70% patients are discovered in an advanced stage.The survival rate of advanced ovarian cancer is less than 30%.However,most patients with early-staged ovarian cancer can be cured or have complete remission after surgery and chemotherapy,Whose the 5-year survival rate can up to 90%.In the United States,the researchers find that ovarian cancer is the fifth common death cause of American women in malignant tumors.So,early diagnosis and early treatment are very important for improving the survival rate of ovarian cancer patients.The lack of specific clinical manifestation and sensitive screening method in early stage is one of the difficulties for the diagnosis and treatment of ovarian cancer,so studying the molecular mechanism for the initiation and development of ovarian cancer is very necessary to look for new targets for early diagnosis and treatment of ovarian cancer.Recent studies show that TCF21 is a tumor suppressor gene with a low expressive status among a variety of tumors,such as head and neck cancer,lung cancer,breast cancer,colon cancer,stomach cancer,melanoma,and so on.There are two aspects about mechanism of TCF21 in tumor: one is that promoter methylation lead to down-regulation of expression which have been confirmed by many studies,and the another is that Zhang etc.found TCF21 can be down-regulated by miR-21 in kidney cancer to inhibit the expression of Kiss-1 gene and increase the invasion ability of kidney cancer,showing that mi R-21 may be the upstream regulating gene of TCF21 while Kiss-1 may be the downstream regulating gene,the studies have fund that Kiss-1 may be the downstream regulating gene of TCF21 in malignant melanomas,kidney cancer and colorectal cancer.Whether TCF21 only regulate Kiss-1 expression to inhibit the transform of the tumor or has other downstream genes to regulate the proliferation and apoptosis remains further study.Its fund that the loss of heterozygosity at 6q22 and 6q23-q24 has been associated with malignant melanomas,lung,head and neck,breast and ovarian tumors.TCF21 gene just locate at 6q23-q24 and has been proved has a low expressive status among malignant melanomas,lung,head and neck and breast tumors.However,there is no literature reports about the expression and mechanism of TCF21 in ovarian tumor at present.Objective: This study investigates the function of TCF21 on proliferation and apoptosis in ovarian cancer cells,and mechanism of TCF21 in tumor.Methods:1 The mRNA and protein expression levels of TCF21 were assessed by fluorescence real-time quantitative PCR and Western-blot methods in ovarian cancer tissues and normal ovarian tissues respectively.2 The Gateway cloning technology was used to clone the target gene TCF21 onto the destination vector,then the destination vector was detected by Agarose gel electrophoresis and DNA sequencing.3 Lentiviral transfection technology was used to get highly expressing TCF21 gene in the ovarian cancer Ovcar8 cells,then the transfected cells was sscreened with antibiotic.4 The cells were divided into two groups: control group(Ovcar8)and test group(Ovcar8-TCF21),then the expression levels of TCF21 were assessed by fluorescence real-time quantitative PCR and Western-blot methods in the 1th,3th,5th generation cells of the test group and the control group.5 MTT method tested the effect of TCF21 high expression on the Ovcar8 proliferation.6 Flow cytometry tested the influence of TCF21 high expression on the Ovcar8 apoptosis.7 The expression levels of CyclinA,B,C,D and CKD2,3,4,6 were detected by fluorescence real-time quantitative PCR in the test group and the control group respectively.Results: 1 The expression level of TCF21 mRNA in ovarian cancer tissues: Both Fluorescence real-time quantitative PCR and Western-blot show that the TCF21 mRNA and protein expression levels in ovarian cancer tissues are lower than in normal ovarian tissue;2 Testing of destination vector: electrophoresis banding displays two srip,target gene DNA sequence test shows that the sequences of destination vector match the 69-1028 bp of TCF21 gene;3 Observation of the transfection efficiency: The antibiotics screening show that all cells in the control group died while 90% cells in the test group survive;4 The expression of target genes: Fluorescence real-time quantitative PCR and Western-blot show that the TCF21 mRNA and protein expression levels of the 1th,3th,5th generation cells in the test group are higher than the control group;5 MTT method detected the influence of TCF21 high expression on the Ovcar8 proliferation capacity: Comparing the test group with the control group,the OD values have no significant difference in 24 h,while the OD values in the test group are lower than the control group in 48 h,72h,96 h with a statistically significant differences(P<0.05);6 Flow cytometry detected the influence of TCF21 high expression on the Ovcar8 apoptosis: The cell apoptosis rate in the test group is significantly increased than in the control group with a statistically significant difference(P<0.05);7 The expression level of CyclinA,B,C,D and CKD2 3,4,6 in the test group and the control group: Fluorescence real-time quantitative PCR shows that the expression level of CyclinC and CKD2 in the tast group is significantly reduced than in the control group with a statistically significant difference(P<0.05),while there is no significant difference in other genes.Conclusion:1 In ovarian cancer tissue,the expression of TCF21 gene is reduced.2 After the high expression of TCF21,the cell growth of Ovcar8 is inhibited and apoptosis rate increases.