Identifying The Mechanism Of The Interaction Of ELL2 And P53 On DNA Repair In Prostate Cancer

Background and aims:Prostate cancer is one of the most common malignancies in male reproductive system.Radiotherapy and chemotherapyare important methods for the treatment of prostate cancer.However,when the disease progression approach to castration-resistant prostate cancer state(CRPC),the tumor will be resistant to radiotherapy or chemotherapy,resulting in tumor recurrence and metastasis,which has always been a major problem for clinicians.Therefore,it is of great clinical significance to introduce various sensitization schemes of radiotherapy and chemotherapy to improve the sensitivity of malignancies and enhance the effect of radiotherapy and chemotherapy.In the preliminary study,our team has found that there is a close relationship between ELL2 and DNA damage.A number of studies have shown that ELL2 is a tumor suppressor gene for prostate cancer.Under the action of DNA damage and other stimulations,transcription factor p53 can induce a variety of responses,including DNA repair,cell cycle arrest and apoptosis,which make p53 an undisputed tumor suppressor.This study aims to clarify the interaction between ELL2 and p53 and their effects on the chemotherapy sensitivity of prostate cancer cells,and further explore the related mechanism from the point of view of DNA damage repair,so as to provide new ideas,new strategies and new targets for the clinical treatment of prostate cancer.Methods:1.We select hormone independent human prostate cancer cell line C4-2 and human embryonic kidney cell line HEK293T.The former was not transferred into plasmids,while the latter was infected with constructed Flag-ELL2 and GFP-p53 plasmids and control plasmids Flag vector or GFP vector.The former used agarose beads coupled with p53 antibody to carry out endogenous Co-IP study,while the latter used agarose beads coupled with Flag or GFP antibody to carry out exogenous Co-IP study(Co-immunoprecipitation).The protein that can react to Co-immunoprecipitation was verified by western blot.2.We prepared two copies of C4-2 and HEK293T cells in parallel for 24 hours,and then transfected into two plasmids GFP vector,RFP vector,GFP-ELL2 or RFP-p53.Then we use immunofluorescence techniqueto verify the co-localization of p53 and ELL2.3.We constructed plasmids that containing different domains of p53 with EGFPcl as skeleton.After successful construction,plasmids containing different domains of p53 and Flag-ELL2 plasmids were transfected into HEK293T cells.After 48 hours of culture,exogenous Co-IP experiments were used to study which domain of p53 can form immune complexes with ELL2,that is,the region of interaction.4.We prepared two groups of C4-2 cells in parallel.After 48 hours of siRNA transfection,one group was treated with 0.4μg/mL Dx(doxorubicin)to induce DNA double-strand damage,and the other group served as a blank control group.Each group was divided into 4 small groups and transfected respectively with plasmids siCtrl,sip53,siELL2,sip53 and siELL2(siDouble).The number and size of the colony formation of tumor cells was collected and compared to analyze the proliferation ability.The cell viability of different treatment groups was investigated by MTT experiment,and the corresponding apoptosis of different treatment groups was detected by flow cytometry.5.After transfection of siELL2 and/or sip53 into different prostate cancer cells,two different prostate cancer cell lines(C4-2 cells and LNCaP cells)were induced respectively by Dx and y-rays.Western blot was used to detect the expression of y-H2AX in different treatment groups.Comet tail distance was detected by comet assay to determine the impact of ELL2 and p53 in DNA damage.6.The plasmids siCtrl and siELL2 were transfected into prostate cancer cells respectively,and then add different concentrations of Dx to induce different degrees of DNA damage.The expression of p53,p-p53(Ser15),p21,CDK2 and Cyclin E was detected by western blot.The result was analyzed to describe the interaction between ELL2 and p53 and their role in the repair of DNA damage in prostate cancer cells.Results:1.Exogenous Co-IP studies on HEK293T cells showed that Flag antibody coupled agarose beads can precipitate Flag-ELL2,and GFP-p53 can also be precipitated;Flag-ELL2 was precipitated when GFP-p53 was precipitated by agarose beads coupled with GFP antibodies;In the endogenous Co-IP study of C4-2 cells,the agarose beads coupled with p53 antibody could precipitate not only p53 but also ELL2 at the same time.2.In the study of cell co-localization,only when the plasmids labeled ELL2 or p53 were transfected,the fluorescence localization in the nucleus could be observed under the laser confocal microscope.and the spotted nucleus localization formed when the GFP-ELL2 and RFP-p53 plasmids were transfected into the cells at the same time.This is different from the location of the diffusion nucleus after transfection respectively of the above two plasmids,and there is obvious co-localization between the two plasmids.3.The results of exogenous Co-IP assay of different domains of ELL2 and p53 showed that only GFP-p53-DBD could bind stably to Flag-ELL2,while GFP-p53-TAD and GFP-p53-CT domains did not react in Co-immunoprecipitation with Flag-ELL2.4.Cell colony formation trials showed that the proliferation of C4-2 cells,that down-regulation of p53 or ELL2 alone and without DNA injury,was significantly higher,but the proliferation of C4-2 cells was more visible when p53 and ELL2 were down-regulated at the same time.In addition,the colony size of C4-2 cells in sip53 group,siELL2 group and siDouble group was larger than siCtrl group.When Dx was added to induce DNA damage in C4-2 cells,the number of colonies in siDouble group was obviously lower than that in siCtrl group.5.After DNA injury induced by Dx,the expression of yH2AX increased after down-regulation of ELL2,while the expression of yH2AX decreased after up-regulation of ELL2.After down-regulation of p53,the expression of yH2AX increased.yH2AX expression increased stronger when the down-regulation of p53 and ELL2 together,compared with down-regulation of p53 or ELL2 alone.6.In C4-2 cells that DNA damage induced by y-ray irradiation,when the expression of ELL2 or p53 was down-regulated alone,the comet tail distance was significantly longer than the control group,and the expression of yH2AX was also higher than the control group.When both p53 and ELL2 were down-regulated at the same time,the extension of comet tail distance was the most in these groups,and the expression of γ-H2AX was the most,too.7.After DNA damage induced by Dx,the expression of ELL2 was down-regulated in prostate cancer cells,resulting in a decrease in the expression of p-p53(Ser15)and p21,while the change of CDK2 and Cyclin E not significant.Conclusion:1.The interaction between ELL2 and p53 can be realized through the interaction of DBD region(DNA bind domain)between ELL2 and the p53..2.Down-regulation of ELL2 and/or p53 not only increased the proliferation of prostate cancer cells,but also increased the sensitivity to chemotherapeutic drugs.And p53 and ELL2 play synergy roles in these two biological effects.3.ELL2 can reduce DNA damage that induced by Dx in prostate cancer cells.4.ELL2 and p53 have synergistic protective effects on DNA damage induced by Dx and y-rays in prostate cancer cells.5.Down-regulation of ELL2 expression can decrease the expression of p-p53(Ser15)and p21 in p53 signal pathway.