The Effect Of MCM2 Protein Phosphorylation Site S139 On The Proliferation Of Ovarian Cancer And Its Mechanism

Objective:Tumor development begins with abnormal cell proliferation,which is caused by abnormal cell cycle.Therefore,the regulatory mechanism of accurate DNA replication and normal cell differentiation is crucial to tumor development.The regulatory mechanism of cell cycle is complex,involving multiple genes and proteins.Among them,MCM is an important promoter of DNA replication,which is necessary for DNA replication in all eukaryotic cells.There are 6 members of MCM family proteins,MCM2?7.MCM2,as a member of its family,has very little MCM2 content in quiescent cells.In proliferating and transforming cells,MCM2 content begins to increase in G₀phase,reaches its peak in late G₀phase and early S phase,and binds to chromatin.It is free MCM2 in s phase to M phase,and decreases in G₂phase and M phase.Because of this cyclical change consistent with cell proliferation,it has been considered as a marker of S-phase cells and as a precancerous marker.Previous studies have found that the phosphorylation site Ser139 of MCM2 protein is significantly different in high and low lymph node metastatic tumor cells,and preliminarily indicated that the phosphorylation site Ser139 of MCM2 plays a role in ovarian cancer metastasis.However,as a marker of tumor cell proliferation,the biological function of MCM2phosphorylation site Ser139 in tumor proliferation has not been fully identified.In this study,we aimed to explore the role of MCM2 phosphorylation site Ser139 in ovarian cancer proliferation and to conduct preliminary studies on its molecular mechanism.Method:1.The database was used to predict the difference of MCM2 expression in ovarian cancer tissues and adjacent tissues and the relationship between high and low MCM2 expression and progression-free survival of ovarian cancer patients.2.To investigate the role of MCM2 in ovarian cancer cells,the no-load pc DNA3.1,pc DNA3.1-MCM2-S139A,and pc DNA3.1-MCM2-S139A were transferred into ovarian cancer HO8910PM cells using transient transfection technique,a.Changes in the number of proliferating cells after phosphorylation of S139 at MCM2 site were detected by EDU-488 cell proliferation assay.b.After phosphorylation of the MCM2site S139 using a plate cloning experiment,the cell clone formation ability was changed.c.Changes in ovarian cancer cell cycle after phosphorylation at the MCM2 locus S139by flow cytometry.d.Changes in cell cycle-related proteins after phosphorylation of MCM2 locus S139 using western blotting.3.Changes in proliferation-related signaling pathways after phosphorylation of MCM2 locus S139 using western blotting.4.After AKT inhibitor MYE-354 treated cells,a.EDU-488 cell proliferation test examined the changes in the expression of proliferative cells after phosphorylation of MCM2 locus S139.b.Plate cloning experiments detected changes in cell clone formation ability after phosphorylation of MCM2 locus S139.c.Changes in ovarian cancer cell cycle after phosphorylation at the MCM2 locus S139 by flow cytometry.5.After treatment with AKT inhibitor MYE354,Western Blotting was used to detect the effect of phosphorylation at MCM2 site S139 on key proteins related to the AKT pathway.After treatment with ERK inhibitor U0126-Et OH,Western Blotting was used to detect the effect of MCM2 site S139 phosphorylation on key proteins related to the ERK pathway.Results:1.The results of the database showed that MCM2 was highly expressed in ovarian cancer tissues and less expressed in paracancer tissues,showing significant differences between the two with statistical significance;There was a statistically significant difference in progression-free survival between ovarian cancer patients with high MCM2 expression and ovarian cancer patients with low MCM2 expression.2.The proliferation detection results of EDU-488 cells showed that the number of proliferating cells increased after the high expression of MCM2,while the number of proliferating cells decreased after the high expression of MCM2-S139A.Compared with the group with high expression of MCM2,the number of proliferating cells in the MCM2 group treated with the AKT inhibitor MYE-354 decreased.3.The results of the plate cloning experiment showed that after the high expression of MCM2,the number of cell clones formed increased and the size of the clones increased.However,after the mutation of the high expression of MCM2-S139A,the number of cell clones formed decreased and the size of the clones decreased,compared with the former.In the MCM2 group treated with the AKT inhibitor MYE-354,the number of cell clones formed decreased and the size of the clones decreased,compared with the MCM2-highly expressed group.4.Flow cell cycle experiment showed that after high expression of MCM2,the proportion of cells in G₀/G₁phase decreased,the proportion of cells in S phase increased,and the proportion of cells in G₂/M phase did not change significantly.However,after site mutation of group MCM2-S139A,the proportion of cells in G₀/G₁phase increased,while the proportion of cells in S phase decreased.Compared with MCM2 group with high expression,MCM2 group cells treated with AKT inhibitor MYE-354 had an increased proportion of G₀/G₁phase cells and a decreased proportion of S phase cells.5.Western Blotting results showed that PI3K,p-AKT?Ser473?,p-ERK?Thr202/Tyr204?and CDK6 were up-regulated and P53,P21 cip1,P27 kip1,Wee1 and Myt1 were down-regulated after overexpression of MCM2.PI3K,p-AKT?Ser473?,p-ERK?Thr202/Tyr204?and CDK6 expression were down-regulated and P53,P21 cip1,P27kip1,Wee1 and Myt1expression were up-regulated after mutations at the MCM2phosphorylation site S139.Compared with the high-expression MCM2 group,p-AKT?Ser473?expression was down-regulated and P53 and P21 cip1 expression were up-regulated in the MCM2 group treated with the AKT inhibitor MYE-354.p-ERK?Thr202/Tyr204?and p-AKT?Ser473?were down-regulated in the MCM2 group treated with the ERK inhibitor U0126-Et OH,compared to the high-expression MCM2 group.Conclusion:We found that MCM2 phosphorylation site S139 promotes the proliferation of ovarian cancer cells and may be involved in the regulation of this biological behavior through the PI3K-AKTt signaling pathway.