The Role And Mechanism Of HELQ In Platinum-Resistance Of Ovarian Cancer

Objective:Ovarian cancer is the most fatal gynecological malignancy and high-grade serous ovarian cancer(HGSOC)is the most common subtype comprising 75% of cases.70% of patients were diagnosed at advanced stages with extensive pelvic-abdominal metastases and massive ascites.Cytoreductive surgery combined with platinum-based chemotherapy is the first-line treatment of ovarian cancer.However,about 20% of ovarian cancer patients are innately resistant to platinum drugs,which ultimately lead to treatment failure.Even 70% of patients who are platinum-sensitive on initial therapy will relapse within 2 years and eventually develop platinum resistance after multiple lines of therapy,often with poor prognosis.Therefore,it is of great clinical significance to find biomarkers that specifically predict platinum resistance in ovarian cancer,identifying patients with platinum resistance early,and explore the mechanism of platinum resistance in ovarian cancer.POLQ-like helicase(HELQ),a 3’-5’ DNA helicase,is involved in the process of DNA replication and damage repair,and is associated with platinum resistance in ovarian cancer.This study explored the application of HELQ in identifying patients with platinum-resistant ovarian cancer and the role and mechanism of HELQ in platinum-resistant ovarian cancer.Methods:1.To elucidate the correlation of HELQ expression in ascites tumor cells with platinum resistance and prognosis in HGSOC patients.(1)The clinical information of 92 newly diagnosed HGSOC patients from January 2014 to September 2019 were retrospectively analyzed,including 60 in the training cohort and 32 in the validation cohort.The expression of HELQ protein in the paired ascites tumor cells and primary tumor tissues were determined by immunohistochemistry.(2)Kaplan-Meier analysis was used to investigate the association between the expression of HELQ in ascites tumor cells and prognosis..(3)Chi-square test was used to analyze the correlation between HELQ expression in ascites tumor cells and clinicopathological characteristics.2.To explore the effect of HELQ expression on the sensitivity of ovarian cancer cells to platinum drugs.(1)Hey A8 Flag-HELQ and A2780 sh HELQ stable cell lines were constructed by lentiviral infection,and verified by RT-PCR and WB.(2)The sensitivity of different ovarian cell lines to cisplatin and other DNA-damaging drugs were detected by CCK8.(3)The effect of overexpression/knockdown of HELQ on the formation of γH2AX foci in ovarian cancer cells treated with cisplatin was determined by immunofluorescence.3.To explore the molecular mechanism of HELQ mediating platinum resistance in ovarian cancer.(1)RNA-Sequence analysis was performed on Hey A8 Flag-HELQ cell.The results were subjected to gene set enrichment analysis and differentially expressed gene screening,and verified by RT-PCR.(2)The relationship between PARP1 and HELQ expression and prognosis in EOC patients was analyzed by online database.The effect of overexpression/knockdown of HELQ on the expression of PARP1 in ovarian cancer cells was detected by WB.The effect of knockdown of PARP1 on cisplatin sensitivity in HELQ-overexpressing cell was detected by CCK8.Results:1.High expression of HELQ in ascites tumor cells is associated with poor prognosis and platinum resistance in HGSOC.(1)The expression of HELQ in ascites tumor cells and primary tumor tissues were positively correlated(R = 0.3135,P = 0.0147).(2)The median PFS of the HELQ low-expression group and high-expression group was 10.6 and 20.9 months,respectively,P = 0.001.The 5-year survival rates were 59% and 73%,respectively,P = 0.027.(3)The incidence of platinum resistance in the HELQ low-and high-expression group was 6.67% and 60%,respectively,P < 0.001.(4)In the validation cohort,the incidence of platinum resistance in the HELQ low-and high-expression group was 11.5% and 66.7%,respectively,P < 0.001.2.HELQ promotes resistance to DNA-damaging drugs of ovarian cancer cells.(1)Compared with Hey A8 Flag(IC 50: 9.556 μM),the sensitivity of Hey A8 Flag-HELQ(IC 50: 18.235 μM)to cisplatin decreased.Compared with A2780 sh Con(IC 50: 22.253 μM),A2780 sh HELQ#1(IC 50:10.135 μM)and A2780 sh HELQ#2(IC 50: 11.345 μM)were more sensitive to cisplatin.(2)Overexpression of HELQ in Hey A8 cells reduced their sensitivity to DNA-damaging drugs.(3)Compared with Hey A8 Flag,γH2AX foci formation was reduced in Hey A8 Flag-HELQ after treatment with cisplatin.Compared with A2780 sh Con cells,γH2AX foci formation was increased in A2780 sh HELQ#1 cells after cisplatin treatment.3.HELQ promotes platinum resistance in ovarian cancer cells by enhancing DNA damage repair.(1)RNA-Sequence results showed that overexpression of HELQ up-regulated the expression of genes related to MMR,BER,HR,NER and Fanconi Anemia pathway.RT-PCR confirmed that the expression of LIG1,PARP1,TDG,POLE2,FEN1 and RPA1 genes in the BER pathway was positively correlated with the expression level of HELQ.(2)Database analysis showed that HELQ expression was positively correlated with PARP1 expression in EOC tumor tissues(R = 0.4,P =1.9e-17)and high expression of PARP1 was associated with poor prognosis.The expressions of HELQ and PARP1 were positively correlated in ovarian cancer cells,and knockdown of PARP1 in Hey A8Flag-HELQ cells increased their sensitivity to cisplatin.Conclusion:1.High expression of HELQ in ascites tumor cells can be used as a predictor in platinum-resistant HGSOC.2.HELQ increases the DNA damage repair of ovarian cancer cells and enhances their platinum resistance by up-regulating the expression of PARP1.