Effects And Mechanisms Of Chk1 And P57/kip2 On Cell Cycle In Human Ovarian Cancer SKOV3 And HO8910 Cells

BackgroundMalignant ovarian tumor is one of three tumors of female genital system, which includes 85-90% epithelial ovarian cancer. Epithelial ovarian cancer has the highest mortality of female genital system and 75% patients in newly diagnosed are moderate and advanced. Clinical therapy includes operation and chemotherapy. 5 year survive rate is about 30% and at least 70% patients will recur. Mechanisms of ovarian cancer are not clear. Recent studies showed DNA damage response (DDR) has an important effect on pathogenesis, development and therapy. Dysfunction of DDR is the key factor of cell carcinogenesis. DDR is closely related to tumor drugs resistance and recurrence because it can promote repairing DNA of tumor cells to decrease the sensitivity of tumor cells to chemotherapy drugs. DDR is a complex regulating system including response to DNA damage, promoting DNA repair by blocking tumor cells cycle and regulating apoptosis and aging. Cell cycle regulating is the key function in the DDR. To further explore the mechanisms of cell cycle and seek new targets to tumor therapy from it is a highlight of tumor therapy. Cell cycle checkpoint kinase 1 (Chk1)and cyclin-dependent kinase inhibitor p57/kip2 are key regulating factors in cell cycle which take part in proliferation, repair and apoptosis of tumor cells and are related to pathogenesis ,development and metastasis of tumor.ObjectiveTo explore the effects and mechanisms of Chk1 and p57/kip2 on cell cycle in human ovarian cancer SKOV3 and HO8910 cells.MethodsPart 1: Based on the principles of siRNA designment,we designed and synthesized three siRNA sequences targeting Chk l.SKOV3 and HO8910 cells were transfected with siRNAs.mRNAs and proteins of Chkl were detected by RT-PCR and western blot,respectively.The siRNA sequences with more effective knockdown were choosen for the further experiments. Selected Chk1 siRNA sequences transfected SKOV3 and HO8910 cells. After 48h, cell cycle distribution of these two cell lines was measured by FCM and mRNA and protein expression of the cell cycle related genes CyclinA,CyclinD1,CyclinE,CDK2,CDK4,p21,p27,Cdc25A were analyzed by RT-PCR and Western blot respectively. Part 2: plasmid of over-expression p57 was constructed and transfected into SKOV3 and HO8910 cells. After 48h, cell cycle distribution of these two cell lines was measured by FCM and mRNA and protein expression of the cell cycle related genes CyclinA,CyclinD1,CyclinE,CDK2,CDK4,p21,p27,Cdc25A were analyzed by RT-PCR and Western blot respectively. Part 3: the expressions of Chk1 and p57/kip2 in human ovarian serous and mucinous carcinoma tissues were measured by immunohistochemistry.ResultsPart 1: Chk1 siRNA targeted interfered the expressions of Chk1 in human ovarian SKOV3 and HO8910 cells and inhibited the G2/M checkpoint blocking of Chk1, while increased the mRNA and protein expression of the cell cycle related genes Cdc25A, CyclinA, CyclinE and CDK2 and decreased the mRNA and protein expression of p21,p27. However, the mRNA and protein expression of CDK4 and CyclinD1 hasn't be changed.Part 2: after being transfected by over-expression p57 plasmid, the expression of p57 was increased and G1/S checkpoint blocking caused by p57 was increased. Over-expression of p57 also could inhibit the mRNA and protein expression of the cell cycle related genes CyclinA,CyclinD1,CyclinE and CDK2. However, the mRNA and protein expression of CDK4, p21, p27 and Cdc25A were not changed by p57 over expression. Part 3: positive rate of Chk1 in human epithelial ovarian cancer was 89.13%(41/46)which was significantly lower than benign and borderline epithelial ovarian tumors(P<0.05). Positive rate of p57 in human epithelial ovarian cancer was 36.96%(17/46)which was significantly higher than benign and borderline epithelial ovarian tumors(P<0.05).Conclusion1. Chk1 could block G2/M checkpoint in human ovarian cancer SKOV3 and HO8910 cells. Targeted inhibiting Chk1 could up-regulate the mRNA and protein expression of the cell cycle related genes CyclinA,CyclinE,Cdc25A,and CDK2, down-regulate the mRNA and protein expression of the cell cycle related genes p21,p27.2. p57 could block G1/S checkpoint in human ovarian cancer SKOV3 and HO8910 cells. Over-expression p57 could inhibit the mRNA and protein expression of the cell cycle related genes CyclinA,CyclinD1,CyclinE,CDK2.3. In human epithelial ovarian cancer, the expression of Chk1 was significantly higher than benign and borderline tumors while the expression of p57 was significantly lower than benign and borderline tumors.