Study On The Mechanism Of PUF60 Promoting The Development Of Ovarian Cancer And The Effectiveness Of Its Small Molecule Inhibitors

Objectives Ovarian cancer(OC)is the second leading cause of gynecologic cancer-related death in women around the world with 5-year survival rate remains below 45%.OC patients are often diagnosed at an advanced stage with unclear etiology and pathogenesis.Therefore,it is urgent to characterize the mechanisms and to discovery a new strategy for OC therapy.One of the genomic characteristics of the OC is copy number alteration(CNA).8q24.3 is the most obvious amplification region in OC,and its amplification predicts poor prognosis.In our previous study,all the genes located in 8q24.3 region were analyzed in multiple databases and found that the splicing factor PUF60 is highly expressed in OC and its high expression predicts poor prognosis,which caught our attention.The purpose of this study was to analyze the expression and function of PUF60 and explore the molecular mechanism of PUF60 during the development of ovarian cancer,to screen out highly effective small molecule inhibitors targeting PUF60 and verify their effectiveness.This study may provide a new idea for OC treatment.Methods(1)Multiple databases were used to analyze the expression and prognosis of PUF60 in OC;the relationship between PUF60 expression and clinical pathological parameters in clinical OC chips was analyzed by IHC.(2)We used the CCK8 cell viability assay and transwell cell migration assay to test the effects of PUF60 on proliferation and migration of OC cells in vitro and established subcutaneous xenograft and lung metastasis models to test the effects of PUF60 on the growth and lung metastasis respectively in vivo.(3)To determine whether PUF60 is a new m6A binding protein by RIP-seq,RNA-pull down and subsequent silver staining and western blot experiments.(4)Through comprehensive bioinformatics analysis of RIPseq,RNA-seq,and m6A-seq data to find target genes regulated by PUF60 and functional classification and signaling pathways of target genes.(5)Verify whether PUF60 can affects the ability of oxidative phosphorylation in OC cells by Seahorse experiment.(6)The effect of PUF60 on the mRNA stability of target genes was detected by RT-PCR.(7)Co-immunoprecipitation(CO-IP)and immunofluorescence(IF)was used to detect the PUF60 interacting protein;IF localization and correlation analysis was used to detect specific ways that PUF60 promotes mRNA decay.(8)Through the virtual and expanded screening of small molecule inhibitors targeting PUF60 to find small molecule inhibitors for further effectiveness verification.(9)Determine the appropriate concentration and half inhibitory concentration(IC50)of small molecule inhibitors in OC cells by the CCK8 method.Detect the binding affinity of the small molecule inhibitor to the PUF60 protein by the surface plasmon resonance(SPR)method.The effect on PUF60 protein expression in OC cells was verified by western blot.(10)The PUF60 expression and the IC50 value of different drug-resistant samples were measured.The effect of small molecule inhibitors on drug-resistant OC cell lines was observed.(11)The effect of small molecule inhibitors on the colony formation in OC cells was verified by cell clone formation experiments.The effect of small molecule inhibitors on the mRNA stability of target genes was detected by RTPCR of actinomycin-treated cells.(12)Constructing a subcutaneous xenograft model and selecting the small molecule inhibitor with the strongest inhibition ability,highest specificity and highest affinity for in vivo effectiveness verification.Results(1)Multiple databases and IHC results show that PUF60 is highly expressed in OC cells,its high expression predicts poor prognosis(P=0.019)and the expression of PUF60 was related to stage and cancer type(Pstage=0.022?Ptype<0.001).(2)In vitro and in vivo experiments show that high PUF60 expression promote the proliferation and migration in ovarian cancer cells.(3)RIP-seq results showed that PUF60 can preferentially bind to m6A general sequence "GGACU",RNA-pull down experiments and subsequent silver staining and western blot experiments showed that the binding affinity of PUF60 protein for the methylated probe(ss-m6A)was significantly higher than that for the unmethylated control(ss-A)(4)Comprehensive analysis of RIP-seq,RNA-seq,and m6A-seq experimental data found that PUF60 target genes were obviously enrichment in oxidative phosphorylation pathway(5)Seahorse experiment results show that PUF60 reduced oxidative phosphorylation and increase glycolysis in ovarian cancer cells.(6)mRNA stability assay showed that PUF60 can promote the mRNA decay of target genes related to oxidative phosphorylation.(7)The results of CO-IP and IF experiments showed that PUF60 interact PABPC1,PUF60 and PABPCl were significantly co-localized to P-bodies and promote the formation of Pbodies in ovarian cancer cells.(8)Preliminary virtual screening of small molecule compounds targeting PUF60 protein showed that GW9508 and ZK 756326 were potential small molecule inhibitors of PUF60.The results of SPR experiments showed that the equilibrium dissociation constants(KD)of GW9508,ZK 756326 were about 66.21 ?M and 75.01 ?M,respectively;They had a concentration-dependent effect on the protein expression of PUF60;They could inhibit the Effects of PUF60 on mRNA stability.(9)Based on analysis of molecular structures of GW9508 and ZK 756326,we optimized and screened 20 analogs.The IC50 values of QL-488B in ES-2 and OVCAR8 with high PUF60 expression were 9.59 ?M;The IC50 values in OVCAR5 and OVCAR3 with low PUF60 expression were 245 ?M and 262 ?j,M,and the IC50 in normal ovarian epithelium cell was 129 ?M.The KD of QL-448B and PUF60 protein was 0.38 ?M;QL-448B versus PUF60 protein show concentration dependent;(10)PUF60 is highly expressed in paclitaxel-resistant and cisplatin-resistant ovarian cancer cells.IC50 of QL-448B in paclitaxel-resistant and non-resistant control cells is 12.99?M and 93.68 ?M,respectively.The IC50 in drug-resistant and non-resistant control cells were 0.37 ?M and 37.35 ?M,respectively.QL-448B was more sensitive to chemotherapy-resistant samples;(11)Cell clone formation experiments showed that QL-448B reduced the ability to form clones,and mRNA stability assays showed that QL-448B inhibited the effect of PUF60 on the mRNA stability of target gene.(12)Nude mouse subcutaneous model showed that QL-448B can significantly reduce the size and weight.The 50mg/kg group had stronger inhibitory effect on the volume(P1omg/kg=0.017?P 50mg/kg=0.0004)and weight(P 50mg/kg=0.0004),indicating that QL448B had a good concentration dependence.Conclusions(1)PUF60 has the potential to serve as an indicator of the poor prognosis of ovarian cancer.(2)PUF60 promote the proliferation and migration of ovarian cancer cells.(3)PUF60 was identified as a new m6A binding protein.(4)PUF60 as m6A binding protein can regulate the level of oxidative phosphorylation and glycolysis to meet the energy needs of cancer cells.(5)PUF60,interact with PABPCl,promotes the decay of target genes in P-bodies.(6)The small molecule inhibitor QL448B has good transformation value with strong inhibitory ability high specificity and affinity in ovarian cells.(7)QL-448B is more sensitive to chemotherapy-resistant treatments and is expected to be applied to platinum-and paclitaxel-resistant treatments.