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Objective:Nuclear factor-kappa B(NF-кB)signaling is implicated in both cancer development and inflammation processes,and has been suggested as a possible target in pain treatment.However,the specific role of the p65 subunit of NFкB in bone cancer pain(BCP)remains largely unknown.The present study was designed to investigate the role for NF-кB in regulation of purinergic receptor(P2X3R)plasticity in dorsal root ganglion(DRG)neurons from rats with BCP.Methods:Bone cancer model was established by injection of Walker 256 rat mammary gland carcinoma cells into tibia of female rats and was verified by X-ray imaging,pathology and behavior examinations.Whole-cell patch clamp recordings were used to measure ATP-evoked responses of tibia DRG(L2-L5)neurons from control and tumor cell treated rats.Real-time polymerase chain reaction and western blot analysis were employed to measure expression of P2X3Rs and related genes at mRNA and protein levels.Methylation status of p2x3r gene promoter was examined in DRGs using methylation-specific PCR and bisulfite sequencing.The interaction between p65 and promoter region of p2x3r gene was identified by chromatin immunoprecipitation assay.Immunofluorescence studies were carried out to determine whether P2X3R was co-expressed with p65 in tibia specific DRG neurons.Results: We showed that tumor cell injection produced mechanical and thermal hyperalgesia,which was associated with upregulation of p65 expression in lumber DRGs.Inhibition of NF-кB signaling using pyrrolidinedithiocarbamate significantly attenuated mechanical allodynia and reversed upregulation of P2X3R expression in DRGs in BCP rats.Tumor cell injection potentiated the fast-activating ATP currents in tibia-specific DRG neurons when compared with control group.Treatment with P2X3R inhibitorA317491 markedly attenuated mechanical allodynia in BCP rats,in a dose-dependent fashion.Interestingly,tumor cell injection also led to a significant demethylation of CpG islands in p2x3r gene promoter region while remarkably downregulated expression of DNA methyltransferase 3a and 3b.In addition,tumor cell injection significantly enhanced ability of p65 to bind the promoter region of p2x3r gene.More importantly,intrathecal injection of recombinant lentiviral vectors designated as LV-shNF-кBp65 for gene silencing remarkably attenuated the mechanical allodynia.Conclusions:Our findings suggest that upregulation of P2X3R expression is attributed to p2x3r gene promoter DNA demethylation and p65 activation.The enhanced interaction of p65 and p2x3r gene promoter would contribute to mechanical allodynia in BCP rats.The lentiviral vector derived shRNA approach against NF-кBp65 is a potential strategy for the management of bone cancer pain.