| Objective:Endometrial cancer(EC)is a gynecological malignancy that seriously threatens women’s health.Endometrioid adenocarcinoma(EAC)is the most common type,accounting for 80-90% of EC.The study of its pathogenesis is the core issue in the prevention and treatment of EC.Studies have found that EC is a common tumor with mutations in DNA damage repair-related genes.When DNA damage repair is abnormal,it will lead to genomic instability and tumorigenesis.However,the current research on DNA damage repair in EC focuses on the microsatellite instability caused by mismatch repair gene abnormalities,and the research on other DNA damage repair pathway abnormalities is relatively few.The Fanconi Anemia(FA)pathway is an important signaling pathway for repairing DNA interstrand cross-links(ICLs)and participating in homologous recombination(HR).and platinum-based drug sensitivity play a key role.Therefore,this study explored the expression of Fanconi Anemia group E(FANCE)gene,a key molecule of the upstream core complex of the FA pathway,in EAC and its correlation with clinicopathological factors,and investigated the involvement of FANCE in vitro and in vivo.Regulatory mechanisms of EAC progression.The research will reveal the role of FANCE in the occurrence and development of EAC,and provide a new theoretical basis for the pathogenesis of EAC.Methods:1.Expression detection of FANCE in EC tissues and analysis of clinicopathological factors:(1)The expression of FANCE mRNA in fresh frozen specimens of10 cases of EAC and 10 cases of normal endometrium,8 cases of EAC and adjacent tissue was detected by RT-PCR.(2)Western blotting(WB)method was used to detect the expression of FANCE protein in fresh frozen specimens of 8 cases of EAC and 8cases of normal endometrium,and 10 cases of EAC and adjacent tissue.(3)The expression of FANCE protein in 83 EACs,83 normal endometrium and 30 dysplastic endometrial tissues collected from 2020.8to 2021.8 was detected by immunohistochemistry.(4)The correlation between the expression of FANCE and the clinicopathological factors of patients with EAC was analyzed.2.Regulation of FANCE on DNA damage repair and detection of drug resistance in EC cells:(1)First,constructing EC cells(HEC-1-A and HEC-1-B)that overexpress FANCE(OE-FANCE)with lentiviral vectors and FANCEknockdown(FANCE-KD)cells with small interfering RNA.Confirmation of FANCE expression efficiency by RT-PCR and WB methods.(2)The formation of DNA damage marker γH2AX fluorescent foci in HEC-1-A and HEC-1-B OE-FANCE and NC cells after 5 μM cisplatin intervention for 1 h was detected by γH2AX immunofluorescence assay.Analysis of fluorescence intensity and foci number was performed by Image J software.(3)The comet tail distance and comet rate of HEC-1-A and HEC-1-B OE-FANCE and NC cells before and after the intervention of 5 μM cisplatin and 4 n M bleomycin sulfate were detected by alkaline comet assay.Using CASP software for analysis,the length of the comet tail distance represents the severity of DNA breakage,and the comet rate represents the percentage of comet-forming cells in the total counted cells.(4)The formation of DNA damage marker γH2AX fluorescent foci in HEC-1-A and HEC-1-B overexpressing FANCE and the corresponding NC cells after 5 μM cisplatin intervention for 24 h was detected byγH2AX immunofluorescence assay,and the fluorescence intensity and foci were analyzed by Image J software with quantitative analysis.(5)Sensitive strain HEC-1-AOE-FANCE and resistant strain HEC-1-B FANCE-KD were detected by CCK-8 cytotoxicity assay,which were resistant to cisplatin and mitomycin C(MMC).The changes in the properties were calculated,and the IC50 of each group of cells was calculated.3.Regulation and cycle detection of FANCE on malignant proliferation and invasion of EC cells:(1)After overexpression and knockdown of FANCE,the proliferation ability of HEC-1-A and HEC-1-B cells was detected by CCK-8,EDU and plate cloning experiments.(2)The changes of migration and invasion ability of HEC-1-A and HEC-1-B cells after overexpression and knockdown of FANCE were detected by Transwell assay.(3)The proportion of cell cycle S phase,G1 phase and G2/M phase of HEC-1-A and HEC-1-B OE-FANCE and NC cells was detected by flow cytometry.(4)The HEC-1-A and HEC-1-B cells in the FANCE-overexpressing and NC groups were synchronized in the early S phase of the cell cycle by thymidine double-blocking method,and released into the cell cycle at the same time During the phase,the 0h,2h,4h,6h,8h and 10 h cell proteins were collected,and cyclins(Cyclin A2,Cyclin B1,Cyclin D1,Cyclin E)and cyclin-dependent kinases(CDK4,CDK6)were detected by WB assay.The protein expression of FANCE was analyzed,and the changes of cell cycle phases after overexpression of FANCE were analyzed.(5)The differential gene and pathway enrichment of HEC-1-A OEFANCE versus NC cells was detected by RNA-Seq sequencing.4.Detection of tumor formation in nude mice with HEC-1-A OEFANCE cells:(1)The HEC-1-A OE-FANCE and NC groups consisted of eight 4-week-old female nude mice in each group.Tumor formation was observed after subcutaneous injection of cells.The body weight and tumor length,width and diameter of nude mice were measured every 2days after tumor formation.The nude mice were sacrificed by cervical dislocation,and the subcutaneous tumor was bluntly dissected.The weight,length and width were measured,and the growth curve of the subcutaneous tumor was drawn.(2)The histological morphology of the subcutaneous tumor paraffin section was detected by HE experiment,and the expression of FANCE and ki-67 in the subcutaneous tumor paraffin section was detected by immunohistochemistry.Results:1.Expression of FANCE in EAC and non-cancer tissue samples:(1)The mRNA expression of FANCE in EAC tissue was significantly lower than that in normal endometrial tissue(p<0.05)and paired paracancerous tissue(p<0.05).(2)The expression of FANCE protein in EAC tissue was significantly lower than that in normal endometrial tissue(p<0.05)and paired adjacent tissue(p<0.05).(3)Immunohistochemical results showed that the low expression of FANCE accounted for 50.6%,20%,and 13.3% in EAC,dysplasia endometrial and normal endometrial tissues,respectively.The expression of FANCE was significantly decreased in endometrioid adenocarcinoma(p < 0.05).(4)The low expression of FANCE was significantly correlated with the older age(>55 years)and the poorly differentiated pathological differentiation of endometrioid adenocarcinoma patients.2.Regulation and drug resistance of FANCE on EC cell damage repair:(1)It was confirmed by RT-PCR and WB experiments that HEC-1-A and HEC-1-B cells significantly overexpressed and knocked down FANCE.(2)After 5 μM cisplatin intervention for 1 h,the number and fluorescence signal of γH2AX foci in HEC-1-A and HEC-1-B OEFANCE cells were attenuated,which were significantly lower than those in the control group(p<0.05).(3)The comet experiment showed that there was no obvious comet tail formation in HEC-1-A and HEC-1-B overexpressing FANCE and NC cells after treatment with no drug or ICL agent(5μM cisplatin),and the comet rate was 0;after addition of DNA fragmentation agent(4 n M bleomycin sulfate(BLEO)),HEC-1-A OE-FANCE cells had a comet rate of 100% with a tail distance of 49.2875 and HEC-1-A NCs had a comet rate of 100 %,the tail distance is 96.9477;the comet rate of HEC-1-B OE-FANCE cells is 100%,the tail distance is 42.531,the comet rate of HEC-1-B NC is 100%,the tail distance is 58.635,and the damage DNA fragmentation in OE-FANCE cells is reduced.In cells added with ICL agent and DNA fragmentation agent,HEC-1-A OE-FANCE cells had a comet rate of 100% with a tail distance of 35.9425,and HEC-1-A NC had a comet rate of 100% with a tail distance of 13.5365;the comet rate of HEC-1-B OE-FANCE cells is 100%,the tail distance is 28.7134,the comet rate of HEC-1-B NC is 100%,the tail distance is 15.323.Compared with the cells that only added the cleavage agent,the comet tail distance of the cells pretreated with ICL agent decreased,while the tail distance of the OE-FANCE cells was longer than that of the NC cells,indicating that the activation of the FA pathway after the overexpression of FANCE repaired part of the damage of ICL.This results in less bound ICL and correspondingly longer comet tails.(4)The sensitivity of FANCE-overexpressing HEC-1-A cells to cisplatin and MMC was reduced,while knockdown of FANCE in the resistant strain HEC-1-B FANCE-decreased HEC-1-B cells were more sensitive to cisplatin and MMC.(5)After HEC-1-A and HEC-1-B cells overexpressed FANCE,the expression of γH2AX protein was significantly decreased,and the expression of FA pathway protein FANCD2 and HR pathway protein RAD51 was significantly increased(p<0.05).A and HEC-1-B cells showed opposite trends(p<0.05).3.Regulation and cycle detection of FANCE on malignant proliferation and invasion of EC cells.(1)The results of CCK-8 experiment,EDU experiment and clone formation experiment showed that the proliferation ability of HEC-1-A and HEC-1-B cells after overexpression of FANCE was significantly decreased,while the proliferation ability of EC cells with FANCE knockdown was significantly increased.(2)Transwell experiments showed that the migration and invasion abilities of HEC-1-A and HEC-1-B cells overexpressed FANCE were significantly decreased,while the migration and invasion abilities of knockdown EC cells were significantly increased.(3)The EC cells overexpressing FANCE and NC were all blocked in the early S phase by thymidine double blocking method.After synchronous release,the cell proteins were collected every 2 h,and the changes in the phase of cyclin passing through the cells were detected by WB.The results showed that Compared with the control group,HEC-1-A OE-FANCE and HEC-1-B OE-FANCE cells showed G2/M transition arrest,prolonged S phase and G2 phase,and inhibited mitosis.(4)Analysis of RNA-Seq sequencing results showed that G2/M transition and mitotic pathway were down-regulated in HEC-1-A cells overexpressing FANCE.And multiple signaling pathways were altered:DNA repair pathway,THFα signaling via NFκB pathway,p53 pathway,PI3K-AKT-MTOR signaling pathway were significantly up-regulated,while IL2 STAT5 signaling pathway,NOTCH signaling,KRAS signaling DN pathway and KRAS signaling UP were significantly down-regulated.4.Detection of subcutaneous tumor formation in nude mice with FANCE overexpressing HEC-1-A cells:(1)The growth rate of subcutaneous tumor in HEC-1-A OE-FANCE group was significantly lower than that in NC group,and the volume and weight of subcutaneous tumor were significantly reduced(p<0.05).(2)The immunohistochemical results of FANCE-overexpressing subcutaneous tumors showed that the expression of FANCE was significantly increased,and the expression of ki-67 was significantly decreased.Conclusions:1.The low expression of FANCE gene is related to the occurrence of EAC.The low-level expression of FANCE is more common in EAC patients older than 55 years old and poorly differentiated.2.The low expression of FANCE gene in EC cells caused the low expression of FA pathway protein FANCD2 and HR pathway protein RAD51,and increased DNA damage,resulting in genomic instability.Upregulation of the FA pathway by FANCE overexpression mediates increased drug resistance in EC cells.3.FANCE gene inhibits mitosis and cell proliferation in EC cells by blocking the phase transition of G2/M cycle and prolongation of S phase.36 figures,27 charts,102 references... |
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