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Background: Primary and metastatic cancers that affect bone are frequently associated with severe and intractable pain.Bone cancer pain has been reported with unique mechanisms and is resistant to morphine treatment.Previous studies have indicated that change of μ-opioid receptor(MOR)expression may be involved in the pathogenesis of bone cancer pain.In addition,neuron-restrictive silencer factor(NRSF)has been recently reported to modulate the transcription of MOR gene.The present study elucidates the regulatory mechanisms of MOR and its potential to effect bone cancer pain.Methods: Using a sarcoma inoculated murine model,pain behaviors that represent continuous or breakthrough pain were evaluated.Micro-CT scanning was used to evaluate the extent of bone destruction.Immunofluorescent staining was used to check the expression of NRSF in the dorsal root ganglion(DRG).Reverse Transcription-Polymerase Chain Reaction(RT-PCR)and Western blot analysis were used to quantify expression of NRSF at the transcriptional and translational levels,respectively.Additionally,chromatin immunoprecipitation assays were used to detect NRSF binding to the promoter of MOR.Furthermore,NRSF was genetically knocked out by antisense oligodeoxynucleotide(AS-ODN),and the expression of MOR and the effect of morphine were subsequently analyzed.Results: The results indicated that after sarcoma cells inoculation,signs of ongoing pain(spontaneous pain)as well as movement induced break-through pain(limb use score and weight bearing score)were detected.Micro-CT images showed various degrees of bone destruction such as radiolucent lesion in the epiphysis and erosion of cortical bone.Expression of NRSF is upregulated in the DRG neurons,and the expression of NRSF mRNA is significantly negatively correlated with expression of MOR mRNA.Additionally,chromatin immunoprecipitation analysis revealed that NRSF binding to the neuron-restrictive silencer element within the promoter area of the MOR gene is significantly promoted with a hypoacetylation state of histone H3 and H4.Furthermore,genetically knocking down of NRSF with AS-ODN rescued the expression of MOR.Eventhough AS-ODN treatment did not alter the baseline pain behavior in sarcoma bearing mice,the analgesic effects of intraperitoneally administrated morphine were significantly potentiated by NRSF gene knock down.Discussion and Conclusion: The present results suggest that in sarcoma induced bone cancer pain,NRSF induced downregulation of MOR is involved in the reduction of morphine analgesia.Epigenetically,up-regulation of MOR could substantially improve the effect of system delivery of morphine.The results indicate that NRSF plays an important role in the modulation of MOR transcription and may represent a novel analgesic target for bone cancer pain.What should be noted is that the expression of opioid receptors are not only regulated at the transcriptional level but also controlled by extensive posttranscriptional processing.Further studies are needed at both the preclinical and clinical levels to develop pharmacological therapy,and to effectively block/relieve bone cancer pain with the goal of increasing the functional status and quality of life of humans with bone cancer pain.