A Study On The Role Of MCM2 Phosphory Lation Site S27 In Proliferation,migration And Apoptosis Of Ovarian Cancer

Aim:Ovarian cancer is the third largest tumor in the female reproductive tract after cervical cancer and endometrial cancer.Ovarian cancer is the highest mortality rate in gynecological tumors.Most ovarian cancer patients are already advanced,and advanced ovarian cancer is prone to metastasis.The prognosis of patients with metastatic ovarian cancer is less than 30%.Due to the high mortality of ovarian cancer and these characteristics of ovarian cancer,studies on factors affecting the infiltration and metastasis of ovarian cancer play an important role in the treatment of ovarian cancer.MCM2 is a member of the minichromosome maintenance protein(MCM)family of six different subunits(MCM2-7).MCM2 is a phosphorylated protein with many related phosphorylation sites,which are regulated and catalyzed by the corresponding kinases to regulate the function of MCM2.MCM2 phosphorylation plays an important role in tumor cell proliferation,DNA damage repair.A pre-laboratory study found that the phosphorylation site Ser27 of MCM2,and preliminary indicates that Ser27 is involved in tumor proliferation and metastasis.However,there are many other kinases at the phosphorylation site Ser27 of MCM2 site.This study aims to consummate the kinase profile of MCM2 Ser27 and to clarify the biological function and molecular mechanism of MCM2 phosphorylation deletion mutant S27 A.Provide new ideas for targeted treatment of ovarian cancer.Method:1.Used a kinase prediction database to predict the kinases at the MCM2 Ser27 site.2.Detect the relationship between CDK2 and MCM2.a.Protein immunoprecipitation(Co IP)were used to detect the interaction between CDK2 and MCM2.b.In vitro kinase assay were used to detect whether CDK2 can phosphorylate MCM2.3.MCM2 and MCM2 S27 A high expression cell lines were constructed,and the control group in which CDK2 were inhibited capacity added to detect the change of ovarian cancer cell proliferation ability.a.CCK8 were used to analysis the proliferation of ovarian cancer cells b.Colony formation assays were used to detect changes in clonality of ovarian cancer cells c.Western blot analysis were used to detect changes in partially proliferating proteins.4.MCM2 and MCM2 S27 A high expression cell lines were constructed and a CDK2 inhibited control group was added to detect capacity in ovarian cancer cell EMT.a.The wound-healing scratch assays were performed to test the migration capacity in ovarian cancer cells.b.Transwell experiments were used to analyze capacity of migration and invasion in ovarian cancer cells.c.Western blot analysis were used to test the expression of EMT-related molecular markers.5.Construction of MCM2 and MCM2 S27 A high expression cell lines to test the change of ovarian cancer cell apoptosis capacity.a.DAPI staining assay were used to test the morphological capacity of ovarian cancer cells.b.Flow cytometry were used to test the apoptosis capacity of ovarian cancer cells to.c.Western blot analysis were used to detect the expression changes of some apoptotic molecular markers.6.Western blot analysis were used to detect the changes of some JAK-STAT pathway proteins.Result:1.CDK2 interacts with MCM2 and CDK2 phosphorylates the Ser27 site of MCM2(1)The possible kinases at the MCM2 Ser27 site were predicted using the GPS2 specific site kinase prediction database.The correlation between MCM2 and CDK2 in 426 cancer tissues and 88 adjacent tissues of ovarian cancer was further analyzed by GRPIA database.(2)CoIP showed interaction between MCM2 and CDK2;CDK2 kinase inhibitor PHA-848125 significantly reduced p MCM2-Ser27;in the presence of ATP,CDK2 active kinase cound not make MCM2 mutant S27 A phosphorylation,but could phosphorylate Ser27 of MCM2 and S139A2.CDK2 promotes the proliferation of ovarian cancer cells by phosphorylating MCM2 at Ser27.(1)In the CCK8 experiment,the OD value of the MCM2 inhibitor PHA-848125 treated MCM2 group was significantly lower than that of the MCM2 wild group;in the plate cloning experiment,the CDK2 inhibitor PHA-848125 was treated compared with the wild-type MCM2 group.The number of cell clones in the MCM2 group of the two ovarian cancer cells was significantly reduced.At the same time,in both experiments,whether the mutation group MCM2 S27 A treatment had no significant effect on the results.(2)Inhibitor PHA-848125 treated MCM2 group can significantly up-regulate P53,P21 expression and down-regulate PCNA expression compared with MCM2 wild group.3.CDK2 promotes migration and invasion of ovarian cancer cells by phosphorylating MCM2 at Ser27.(1)In the scratch test and Transwell migration experiment,the inhibitor PHA-848125 reduced the cell migration number of the MCM2 high expression wild group;the Transwell invasion test also showed that the inhibitor PHA-848125 were significantly reduced the number of cell invasions compared with the MCM2 high expression wild group cells.At the same time,in the three experiments,whether the mutation group MCM2 S27 A treatment had no significant effect on the results.(2)Inhibitor PHA-848125 treated MCM2 group can significantly up-regulate the expression of Marker E-cadherin and down-regulate the expression of Vimentin,N-cadherin and ZEB1 compared with MCM2 wild group.4.MCM2 mutant S27 A promotes apoptosis in ovarian cancer cells.(1)MCM2 S27 A group showed more typical apoptotic features,while wild-type MCM2 cells showed less abnormal nuclear changes.In flow cytometry,the MCM2 S27 A group had more cells in the wild-type MCM2 group for early apoptosis.(2)Compared with MCM2 wild type,S27 A mutant down-regulated the expression of BCL-2,BCL-XL,Caspase3 and Caspase9,and up-regulated the expression of BAX,BAD,Cleaved Caspase3,Cleaved Caspase9 and Cleaved PARP.5.Compared with MCM2 wild type,S27 A mutant down-regulated the expression of JAK1,p-JAK1(Y1022+Y1023),JAK2,p-JAK2(Y1007+Y1008),STAT1,p-STAT1(Tyr701),STAT3,STAT3(Tyr705),p-STAT3(Ser727),and p-STAT5(Tyr696).Conclusion:1.CDK2 interacts with MCM2 and CDK2 phosphorylates the Ser27 site of MCM2.2.CDK2 promotes the proliferation of ovarian cancer cells by phosphorylating MCM2 at Ser27.3.CDK2 promotes the metastasis and invasion of ovarian cancer cells by phosphorylating MCM2 at Ser27.4.MCM2 mutant S27 A can promote apoptosis of ovarian cancer cells.5.MCM2 mutant S27 A can inhibit JAK-STAT pathway activation...