The Role And Mechanism Of TRDMT1-mediated DNA Damage Repair In Ovarian Granulosa Cells

ObjectiveTo explore the repair effect and mechanism of TRDMT1 on the DNA damage of granular cells（GCs）caused by premature ovarian failure（POF）.Methods1.Establishing and identifying of POF model in vivo:Cyclophosphamide（CTX）to establish POF in vivo model,intraperitoneal injection of PBS and bone marrow-derived mesenchymal stem cells（BMSCs）to establish POF-PBS and POF-BMSCs model to evaluate the general condition of rats,vaginal estrus cycle,HE staining to observe ovarian structure,tissue electron microscope to observe the ultrastructure of ovarian tissue,ELISA to detect serum hormones,and Tunnel to detect tissue apoptosis.Establishing and identifying POF model in vitro:culturing ovarian granulosa cells KGN in vitro to establishing POF model in vitro,establishing CTX-KGN co-culture system with PBS and BMSCs,flow cytometry to detect the apoptosis of CTX-KGN-PBS and CTX-KGN-BMSCs groups.2.Verifying the correlation between TRDMT1 and POF:immunohistochemi stry,immunoblotting and immunofluorescence to detect the expression of TR DMT1 in POF model and the serum of POF patients3.Exploring the role and mechanism of TRDMT1 in regulating GCs DNA damage repair:after changing the expression level of TRDMT1 in CTX-KGN,probe method to detect ROS level,comet electrophoresis to detect DNA damage,immunofluorescence to detectγ-H₂AX expression,Western Blotting to detect the expression of RAD51,RAD52.Exploring whether TRDMT1-mediated DNA damage repair is related to its methylation:observing the effect of TRDMT1wild-type and mutants(TRDMT1^G155V,TRDMT1^E63K)on the DNA damage repair and m RNA methylation level of CTX-KGN.Results1.POF models in vivo and in vitro models were successfully established:the rats in the POF-PBS group were generally in poor condition,eating activity was reduced,the estrus cycle was disturbed,serum hormones E2 and AMH decreased,FSH and LH increased,atretic follicles increased,and GCs apoptosis increased.The general condition of the rats in the POF-BMSCs group improved,the estrus cycle was restored,the atretic follicles decreased,the serum E2 and AMH increased,the serum FSH and LH levels decreased,and the GCs apoptosis in the ovarian tissue decreased.Successfully established a POF in vitro model:CTX-KGN cell apoptosis increased,and cell apoptosis decreased after co-culture with BMSCs.2.TRDMT1 was related to POF:TRDMT1 decreased in both in vivo and in vitro models of POF.Compared with healthy people with normal ovarian reserve,the expression of serum TRDMT1 in patients with POF decreased.3.TRDMT1 increased the DNA damage repair ability of GCs:after overexpression of TRDMT1,the DNA damage of cells was reduced,and cell apoptosis reduced,and the clearance ofγ-H₂AX was also promoted.After TRDMT1 was knocked down,the opposite trend was shown.4.TRDMT1 increased DNA repair of GCs by down-regulating ROS:Compared with healthy people,the level of ROS in GCs of POF patients increased significantly,and the level of ROS in POF models in vivo and in vitro also increased.The total ROS level of cells increased with the increase of the concentration of H₂O₂,and was accompanied by the increase of DNA damage,the delay of the clearance ofγ-H₂AX foci,and the increase of apoptosis.After H₂O₂treatment of cells,TRDMT1 overexpression reduced DNA damage in KGN cells,and DNA damage increased after TRDMT1 was down-regulated.TRDMT1inhibition also delayed the clearance ofγ-H₂AX foci.N-Acetyl-L-cysteine（NAC）treatment inhibited the generation of ROS in H₂O₂+KGN,and significantly reduced the DNA damage in KGN cells.After TRDMT1 was up-regulated,cellular DNA damage decreased,and the clearance efficiency ofγ-H₂AX foci increased,and the trend was reversed after TRDMT1 was down-regulated.5.TRDMT1-mediated DNA damage repair of GCs was related to its m RNA methylation:G155V mutation lead to decrease in the methylation activity of TRDMT1,while the E63K mutation significantly increased its methylation activity.CTX-KGN after TRDMT1 knockdown-TRDMT^WTand mutant TRDMT1^G155V/E63Kwere stably expressed in KGN.Compared with TRDMT1^WTtransfection,ROS levels and apoptosis of TRDMT1^E63Kgroup were reduced,and the efficiency of DNA damage repair increased.ROS levels,apoptosis and DNA damage of TRDMT1^G155Vgroup increased,and the efficiency drop.ConclusionTRDMT1 reduces the oxidative DNA damage of GCs and increases its repair ability,which is beneficial to the preservation of fertility in patients with ovarian dysfunction caused by chemotherapy.