Font Size: a A A

The Role And Mechanism Of XAB2 In Platinum Resistance Of Ovarian Cancer

Posted on:2024-08-09Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z ChenFull Text:PDF
GTID:1524307310494054Subject:Clinical Medicine
Abstract/Summary:PDF Full Text Request
Objective:Epithelial ovarian cancer(EOC)is the gynecological tumor with the worst prognosis.Numerous studies have demonstrated that the size of residual lesions after ovarian cancer tumor debulking surgery and the tumor’s sensitivity to platinum-based chemotherapy are crucial factors determining the prognosis of ovarian cancer.Approximately 20% of newly diagnosed ovarian cancer patients are insensitive to platinum drugs,leading to treatment failure.Even in ovarian cancer patients who are sensitive to platinum drugs and undergo satisfactory tumor cell debulking surgery and standardized chemotherapy,recurrence still occurs,and eventually,resistance to platinum drugs develops.In-depth exploration of the specific mechanisms underlying platinum resistance in ovarian cancer is of significant importance for prolonging the survival time of ovarian cancer patients.In our previous study,through analysis of the TCGA database,our research team found that patients with platinum resistance exhibited high expression of Xeroderma Pigmentosum group A binding protein 2(XAB2),and the high expression of XAB2 in the ascites of ovarian cancer patients in our hospital was significantly associated with a shortened progression-free survival(PFS)and overall survival(OS).Therefore,this study aims to further explore the molecular mechanisms of XAB2 in platinum resistance in ovarian cancer cells,providing new scientific evidence for the prediction and treatment of platinum resistance in ovarian cancer.Methods:1.Through lentiviral transduction,A2780-sh XAB2,HEY-sh XAB2,and SKOV3-XAB2 stable ovarian cancer cell lines were established with stable knockdown of XAB2 and stable overexpression of XAB2,respectively.The proliferation of these ovarian cancer cells and their IC50 values for cisplatin(DDP)were determined using the CCK-8 cell viability assay.The expression levels of 5-ethynyl-uridine(EU),a uridine analogue,were measured by cell immunofluorescence to observe the recovery of newly transcribed RNA in A2780-sh XAB2 and SKOV3-XAB2 cells.2.Cyclobutane pyrimidine dimers(CPDs)and phosphorylated histone H2AX(γ H2AX)are markers of DNA damage in cells.Cell immunofluorescence was performed at different time points after cisplatin treatment to detect the formation and removal of CPDs and γH2AX in A2780-sh XAB2 and SKOV3-XAB2 cells.Western blotting(WB)and reverse transcription-polymerase chain reaction(RT-PCR)were used to detect the transcription and expression levels of DNA damage repair key factors RNAPⅡ,XPC,and ERCC1 at the same time points in SKOV3-XAB2 cells.3.RNA sequencing was conducted to identify differentially expressed protein-coding genes in A2780-sh XAB2 compared to A2780-NC cells.Downstream genes involved in tumor cell DNA damage repair were screened,and their expression levels in A2780-sh XAB2 and A2780-NC cells were validated using WB and RT-PCR.Analysis of the TCGA database was performed to explore the correlation between candidate genes HMGA2 and XAB2,as well as the expression of HMGA2 in tumor tissues from ovarian cancer patients.Additionally,under cisplatin treatment,the expression of HMGA2 in A2780-sh XAB2 was detected by RT-PCR.Results:1.High expression of XAB2 promotes platinum resistance in ovarian cancer cells.(1)A2780-sh XAB2,HEY-sh XAB2,and SKOV3-XAB2 ovarian cancer cell lines were successfully established with XAB2 knockdown and overexpression.Compared to the control group,high expression of XAB2 promoted the proliferation of ovarian cancer cells,while XAB2 knockdown inhibited cell proliferation.After 48 or 72 hours of cisplatin treatment,SKOV3-XAB2 cells showed a higher IC50 for cisplatin compared to SKOV3-NC cells(P<0.05),while A2780-sh XAB2 cells exhibited a lower IC50 for cisplatin compared to A2780-NC cells(P<0.05).Similarly,HEY-sh XAB2 cells had a lower IC50 for cisplatin compared to HEY-NC cells(P<0.05).(2)Under cisplatin treatment,XAB2 promoted the transcription of newly synthesized RNA in ovarian cancer cells.Compared to SKOV3-NC cells,SKOV3-XAB2 cells exhibited higher expression of EU after cisplatin treatment and cisplatin withdrawal(P<0.001),while A2780-sh XAB2 cells had lower expression of EU after drug withdrawal compared to A2780-NC cells(P<0.01).2.XAB2 is involved in different DNA damage repair pathways in ovarian cancer cells.(1)XAB2 promotes nucleotide excision repair(NER)of DNA damage in ovarian cancer cells under cisplatin treatment.Immunofluorescence analysis showed that the expression of CPDs in SKOV3-XAB2 cells was lower than in the control group during cisplatin treatment and drug withdrawal(P<0.01),while the expression of CPDs in A2780-sh XAB2 cells was higher than in the control group(P<0.05).WB and RT-PCR confirmed the upregulation of key factors RNAPII and ERCC1 in the transcription-coupled repair(TCR)pathway in SKOV3-XAB2 cells,while no significant difference was observed in the expression of XPC,a key factor in the global genome repair(GGR)pathway.(2)XAB2 promotes homologous recombination(HR)DNA damage repair in ovarian cancer cells under cisplatin treatment.Immunofluorescence analysis showed that the expression of γH2AX in SKOV3-XAB2 cells was lower than in the control group during cisplatin treatment and drug withdrawal(P<0.01),while the expression of γH2AX in A2780-sh XAB2 cells was higher than in the control group(P<0.05).3.XAB2 may be involved in homologous recombination repair of DNA in ovarian cancer cells through the regulation of HMGA2.(1)RNA sequencing results showed that HMGA2 is a potential protein-coding gene regulated by XAB2.Validation by RT-PCR and WB in A2780-sh XAB2 cells revealed downregulation of HMGA2 protein and m RNA expression compared to the control group.(2)Analysis of the TCGA database in ovarian cancer patients showed that HMGA2 is highly expressed in tumor tissues compared to normal ovarian tissues(P<0.05),and its expression level is positively correlated with XAB2 expression(P=0.002).Conclusion:1.XAB2 promotes the repair of DNA damage of ovarian cancer cells under the action of platinum drugs through nucleotide-excision repair pathway and homologous recombination pathway.2.HMGA2,the potential regulator of XAB2,is positively correlated with the expression of XAB2.The knockdown of XAB2 significantly inhibits the expression of HMGA2 and PARP1,the key factor of DNA damage repair recognition targeted by the latter.
Keywords/Search Tags:Epithelial ovarian cancer, XAB2, DNA damage repair, Platinum resistance
PDF Full Text Request
Related items