SH3RF2 Affects Malignant Biological Behavior And Cisplatin Resistance Of Ovarian Cancer By Regulating The RBPMS

Background:Ovarian cancer is the leading cause of death in the female reproductive system tumors.The onset of ovarian cancer is insidious and the early symptoms are lack of specificity.Most patients are already advanced when they are diagnosed and even with local spread or distant metastasis,missing the best opportunity for treatment.The overall prognosis of patients with ovarian cancer is poor,especially in advanced patients,with a 5-year survival rate of only 25%-30%.Continuous cell proliferation signals,genomic instability and mutation,induction of angiogenesis,activation of invasion and metastasis,wireless replication potential,avoidance of immune destruction,evasion of growth inhibition,and resistance to apoptosis are the main characteristics of the malignant biological behavior of tumors,which are also the main reasons for the poor prognosis of patients.Ovarian cancer also has tumor heterogeneity,which manifests as differences in cell morphology,surface markers,and chemotherapy resistance among different patients.Platinum drugs are currently used as first-line drugs for the treatment of ovarian cancer.However,with the widespread use of platinum drugs,the incidence of platinum resistance is also gradually increasing.According to statistics,about 30-50%of ovarian cancer patients will develop resistance after receiving platinum drugs.Therefore,it is particularly important to explore how to reverse the malignant biological behavior and platinum resistance of ovarian cancer.In-depth study of the molecular mechanisms regulating the malignant biological behavior and platinum resistance of ovarian cancer can provide an important theoretical basis for finding new treatment strategies.To help improve the survival and prognosis of patients with ovarian cancer.SH3RF2,also known as SH3 Domain Containing Ring Finger 2,belongs to the E3 ubiquitin ligase family and plays a crucial role in various physiological processes.It is involved in cell proliferation,differentiation and regulation of the cell cycle.Additionally,it is implicated in cell migration,invasion,and has a significant impact on tumor development and metastasis.SH3RF2 is a recently discovered tumor suppressor gene,which plays a vital regulatory role in tumor occurrence and development.Mutations and abnormal expression of SH3RF2 are associated with various diseases.For instance,abnormal expression of SH3RF2 is found in many common tumors such as breast cancer,colorectal cancer and prostate cancer,and it is significantly associated with poor prognosis of patients.However,the effect of SH3RF2 on the malignant biological behavior of ovarian cancer and cisplatin resistance has not been reported in the literature.Therefore,the aim of this study is to explore the effect of SH3RF2 on the malignant biological behavior of ovarian cancer cells,such as proliferation,apoptosis,DNA damage.To investigate whether SH3RF2 is associated with cisplatin resistance and explore its molecular mechanism,so as to provide new potential therapeutic targets and theoretical basis for molecular targeted therapy of ovarian cancer.Methods:Part 1.① The expression of SH3RF2 in ovarian cancer tissues and normal ovarian tissues was analyzed based on GEPIA database and TNMplot database.The GSE15709 dataset in the GEO database was used to analyze the differential expression of SH3RF2 in human ovarian cancer cell A2780 and cisplatin-induced platinum resistance A2780 cell line.②Based on the previously established ovarian cancer prospective follow-up cohort(OOPS),A nested case-control study was designed with 1:1 matching(matching conditions included age at diagnosis,FIGO stage,tumor size,metastasis,differentiation and pathological type).A total of 120 cisplatin-sensitive patients and cisplatin-resistant patients were enrolled,and their histopathological sections and relevant clinical information were collected.③The expression level of SH3RF2 in the pathological tissue sections of cisplatin-sensitive(60 cases)and cisplatin-resistant(60 cases)patients was detected by immunohistochemistry,and the expression level of SH3RF2 in the sections was scored according to the nuclear staining intensity semi-quantitative evaluation and the percentage of positive tumor cells.④Chi-square test and Fisher exact test were used to analyze the relationship between SH3RF2 and clinicopathological features(age at diagnosis,FIGO stage,tumor size,metastasis,pathological grade and pathological type)and pathological immunohistochemical markers(PAX8,WT-1,P16,ER,P53,Ki-67,Vimentin and NapsinA).Logistic regression,Spearman correlation and other statistical methods were used to analyze and verify the relationship between SH3RF2 expression level found in the previous public data and cisplatin resistance of patients.Kaplan-Meier survival analysis was used to test the correlation between the expression level of SH3RF2 and the overall survival time of patients.Part 2.① Construction of cisplatin-resistant cell lines:A2780 and SKOV3 cells were treated with gradient concentrations of cisplatin to obtain cisplatin resistant cell lines A2780/DDP and SKOV3/DDP.MTT assay was used to detect the effect of cisplatin on cell viability and IC50 value was calculated.② The mRNA and protein expression levels of SH3RF2 in A2780,SKOV3,A2780/DDP,and SKOV3/DDP cells were detected by qPCR and Western blot.Using lentivirus to construct sh-SH3RF2 stably transfected A2780/DDP and SKOV3/DDP.③ qPCR and Western blot were used to verify the successful transfection of sh-SH3RF2.After incubating the cells with different cisplatin concentrations(0,2.5,5,10,20,40,80 μM DDP)for 48 h,MTT assay was performed to test cell viability and calculate the IC50.Parental,shNC,sh1-SH3RF2,sh2-SH3RF2 groups were treated with 1/2 IC50 concentration of cisplatin for 48 h.Cell apoptosis was detected by flow cytometry and AO-EB staining,cell viability by cloning formation experiment,caspase 3 activity by assay kit,and cellular DNA damage by comet electrophoresis and γ-H2AX immunofluorescence;protein expression levels of cleaved caspase 3,cleaved PARP,γ-H2AX were detected by Western blot.④Cells in logarithmic growth phase were injected subcutaneously into 6-week-old female BALB/c nude mice.When tumor volume reached about 150 mm3,cisplatin or solvent was injected intraperitoneally at a dose of 5 mg/kg(twice a week).Tumor volume was measured every 3 days.After three weeks,mice were euthanized,tumor tissue was collected and photographed.Immunohi stochemi stry was used to detect the expression levels of SH3RF2,Ki-67,cleaved caspase 3 and γ-H2AX in tumor tissues.Part 3.① The Hitpredict and IntA database is utilized for identifying potential downstream target genes having the ability to bind with SH3RF2 and their probable binding sites.Targets with high binding scores are chosen for further validation.②Immunofluorescence is employed to observe the concurrent localization of SH3RF2 and RBPMS within cells.Additionally,a Co-IP approach is utilized to ascertain the binding status between endogenous SH3RF2 and RBPMS in cells.③The expression of RBPMS in pathological tissue sections of ovarian cancer patients included in this nested case-control study was detected by immunohistochemistry,and its relationship with clinicopathological parameters was analyzed.④ Western blot was used to detect the expression level of RBPMS protein in the cells transfected with SH3RF2 shRNA and tumor tissues.After the cells were treated with protein synthesis inhibitor Cycloheximide and proteasome inhibitor MG132,Co-IP and Western blot confirmed that SH3RF2 induced degradation of RBPMS K48 through the proteasome pathway through ubiquitination.⑤A dual-luciferase reporter assay is used to examine the impact of sh-SH3RF2 on the transcriptional activity of AP-1,48 hours post the transfection of A2780/DDP and SKOV3/DDP cells with shNC,sh1-SH3RF2.Concurrently,The expression levels of XIAP and c-Myc mRNA in drug-resistant cells and transplanted tumor tissues were detected by qPCR.⑥ Following si-RBPMS and si-NC transfection into A2780/DDP cells for 24 hours,qPCR and Western blot are used to validate the efficiency of this transfection.Lastly,cells transfected with ShNC+si-NC,sh-SH3RF2+si-NC,sh-SH3RF2+si-RBPMS are subjected to cisplatin treatment for 48 hours.Thereafter,MTT is employed to ascertain cell viability,an assay kit is used to determine caspase-3 activity,flow cytometry is used to detect cell apoptosis,and comet assay together with immunofluorescence is used to identify DNA damage.Results:Part 1.①Based on data from GEPIA and TNMplot databases,SH3RF2 is found to be highly expressed in ovarian cancer tumor tissues compared to normal tissues,and this difference is statistically significant.Furthermore,GEO database shows that SH3RF2 expression levels are found to be elevated in platinum-resistant A2780 cells when compared to platinum-sensitive ovarian cancer cells.②Based on the nested case-control study of OOPS cohort,the above validation analysis results showed that SH3RF2 expression was significantly higher in ovarian cancer tissues of cisplatin-resistant patients than that of cisplatin-sensitive patients after IHC detection of pathological tissue sections of cisplatin-sensitive and resistant patients(χ2=30.00,P<0.001).③ The correlation between the expression level of SH3RF2 in pathological tissue sections and the clinicopathological characteristics of patients was analyzed.The results showed that:The high expression of SH3RF2 was not only closely related to cisplatin resistance in ovarian cancer(χ2=30.00,P<0.001),but also related to tumor size(χ2=4.15,P=0.04),but not related to age,FIGO stage,histological differentiation,pathological subtype,metastasis and related immunohistochemical indicators.Multivariate conditional logistic regression analysis showed that SH3RF2 expression level was associated with cisplatin resistance of ovarian cancer after adjusting for possible confounding factors,and the effect value and 95%confidence interval were 9.96(3.08-32.24).Kaplan-Meier analysis showed that the overall survival time of patients with high SH3RF2 expression was significantly shorter than that of patients with low SH3RF2 expression(χ2=6.20,P=0.013).Part 2.① The MTT experiment results revealed that the IC50 value of the A2780/DDP cell line(61.71 ± 4.10 μM)was significantly higher than that of the wild-type A2780 cell line(10.94 ± 1.27 μM).Similarly,the IC50 value of the SKOV3/DDP cell line was also significantly increased compared to the wild-type SKOV3 cell line,from(9.94 ± 0.95 μM)to(44.89 ± 3.22 μM),indicating the successful establishment of cisplatin-resistant cell lines.② qPCR and Western blot results showed that the mRNA and protein levels of SH3RF2 in A2780/DDP and SKOV3/DDP cells were significantly increased compared to the Parental group.③The results of qPCR and Western blot showed that compared with the shNC group,sh1-SH3RF2 and sh2-SH3RF2 were both able to effectively downregulate the protein expression level of SH3RF2 in A2780/DDP and SKOV3/DDP cells compared to the shNC group,indicating successful SH3RF2 knockdown in cells.In addition,the cell viability was significantly lower,cisplatin IC50 was reduced,and the apoptosis rate was significantly increased in the cell transfected with SH3RF2 shRNA(P<0.05).Compared with DDP+shNC group,DDP+SH3RF2 shRNA group had significantly lower cell viability,increased apoptosis rate,reduced clonogenic survival,and increased expression of cleaved caspase 3 and cleaved PARP.Compared with DDP+shNC group,DDP+SH3RF2 shRNA group exhibited significant comet tail phenomenon,with increased γ-H2AX spots and protein expression.④In vivo studies showed that silencing SH3RF2 significantly inhibited tumor growth and induced apoptosis compared with shNC control group.Compared with DDP+shNC group,DDP+SH3RF2 shRNA group had smaller tumor volume and decreased Ki-67 expression,but increased expression of cleaved caspase-3 andγ-H2AX.Part 3.①The analysis results of Hitpredict database and IntAct database showed that RBPMS had a high interaction with SH3RF2.②Immunofluorescence results show a good co-localization of SH3RF2 and RBPMS in the cell nucleus.Furthermore,Co-IP results further confirm the protein interaction between SH3RF2 and RBPMS.③ IHC showed that the expression of RBPMS in DDP resistant ovarian cancer tissues was significantly lower than that in DDP sensitive tissues.The low expression of RBPMS was closely related to cisplatin resistance of ovarian cancer(χ2=64.53,P<0.001),and was also related to ER positive,but not related to age,FIGO stage,histological differentiation,pathological subtype,tumor size,metastasis and the other pathological immunohistochemical indicators.Multivariate conditional logistic regression analysis showed that RBPMS was associated with cisplatin resistance in ovarian cancer,and the effect size and 95%confidence interval were 0.02(0.00-0.15).Spearman correlation analysis showed that SH3RF2 was negatively correlated with RBPMS.④Western blot showed that the expression levels of RBPMS protein in the cells and tumor tissues of nude mice in the sh-SH3RF2 group were significantly higher than those in the shNC group.Compared with the DDP+shNC group,the expression level of RBPMS protein in the xenograft tumor of nude mice in the DDP+sh-SH3RF2 group was further increased.Co-IP results showed that SH3RF2 could promote the degradation of RBPMS through the proteasome pathway by ubiquitination at K48 site.⑤ Dual-luciferase reporter results show:Compared with the sh NC group,the transcriptional activity of AP-1 was significantly reduced after cells were transfected with SH3RF2 shRNAl and shRNA2.The results of qPCR showed that the expression levels of XIAP and c-Myc mRNA,the downstream target genes of AP-1,were decreased after SH3RF2 silencing in both drug-resistant cells and xenograft tumors in nude mice.Compared with the DDP+shNC group,the expression levels of XIAP and c-Myc mRNA in the DDP+sh-SH3RF2 group were further decreased.⑥Compared with the SH3RF2 shRNA group,co-transfection of si-RBPMS can reduce the sensitivity of A2780/DDP cells to cisplatin,and the cisplatin-induced cell proliferation inhibition,cell apoptosis,and DNA damage functions were all significantly suppressed.Conclusions:1.The upregulation of SH3RF2 is observed in ovarian cancer tissues and cisplatin-resistant specimens,indicating a correlation with cisplatin-resistance,tumor size and unfavorable prognosis in ovarian cancer.2.SH3RF2 plays the role of an oncogene in ovarian cancer.Silencing SH3RF2 can inhibit cell proliferation and promote cell apoptosis,enhance cisplatin-induced cell DNA damage,and increase the sensitivity of tumor cells to cisplatin.3.The expression of RBPMS is decreased in ovarian cancer resistant tissues.SH3RF2 is negatively correlated with RBPMS and highly bound to RBPMS.SH3RF2 can induce the ubiquitination of RBPMS at K48 site,thereby promoting its degradation through the proteasome pathway.4.SH3RF2 can increase the transcriptional activity of AP-1 by inducing the ubiquitination and degradation of RBPMS,affecting the malignant biological behavior and cisplatin-sensitivity of ovarian cancer.