Epigenetic Mechanism Of Purinergic Receptor P2X3R Of Dorsal Root Ganglion In Bone Cancer-induced Pain In Rats

Cancer pain resulting from primary tumors or metastatic carcinoma is one of the most severe and intractable types of chronic pain, which severely compromises quality of life of cancer patients and imposes a huge burden on society. However, mechanism underlying the development of cancer-induced pain(CIP) remains largely unknown, and effective clinical treatment is very limited. To date, no new pharmacotherapy has emerged, so there is an urgent need for new treatments for patients with cancer pain. In recent years, some studies found that tumor and osteoclast induced bone destruction and acidosis causes inflammation, tumor release products and fractures, ischemia, tumor invasion lead to nerve fiber damage may be the main reasons for the formation of bone cancer pain. Purinergic P2 X receptors are preferentially expressed on dorsal root ganglion(DRG) neurons and have been implicated in neuropathic, inflammatory and visceral pain hypersensitivity. However, relatively few studies have been reported on the roles of purinergic P2 X receptors in cancer pain conditions, and the mechanism underlying P2X3 R upregulation under CIP conditions is not fully understood. In the forming process of bone cancer-induced pain, tumor cells and some stromal cells secrete proinflammatory factors to sensitization of peripheral primary afferent neurons, and to increase the expression of inflammation related factors in neurons. NF-κB, an important nuclear transcription factor, is considered to control numerous genes encoding inflammatory and nociceptive mediators. However, whether NF-κB is involved in the formation of bone cancer-induced pain and regulation of P2 X receptor sensitization remains unknown. We will explore the epigenetic mechanism of P2X3 R expression and the function of NF-κB, and provide a theoretical basis for clinical treatment of cancer pain.Part one: To elucidate the role and mechanism of P2X3 R and NF-κB in the formation of bone cancer-induced pain.1. Specific Aims:(1) To establish an animal model of cancer-induced pain and to explore its electrophysiological properties of DRG neurons innervating the bone.(2) To investigate whether P2X3 R involved in the formation of cancer-induced pain.(3) To explore the mechanism of NF-κB involved in cancer-induced pain.2. Methods:(1) Adult female Sprague Dawley(SD) rats were used in the present studies. Bone cancer model was established by injection of Walker 256 rat mammary gland carcinoma cells(4×105) into tibia of female rats and was verified by X-ray imaging, pathology and morphology. Behavior responses to stimulation of von Frey filament and thermal radiation were employed on normal and bone cancer rats. Hindlimb body weight-bearing difference(WBD) was also measured using the incapacitance tester.(2) Di I retrograde tracing method was used to label the tibial specific DRG neurons.(3) Whole-cell patch clamp recordings were performed in vitro on single tibia specific DRG neurons acutely isolated from these rats labeled with dye Di I.(4) Expression of Rel B,p52,p65,p-p65,RANKL,P2X3 R,P2X1R,P2X2 R and H2 S producing enzyme cystathionine-beta-synthetase(CBS) were measured with western blotting and(or) real-time PCR analysis.(5) In order to detect whether P2X3 R is involved in the formation of bone cancer pain, purinergic receptor inhibitor suramin or P2X3 R, P2X2/3R potent inhibitor A317491 were used in the present study.(6) To investigate whether p65 is involved in cancer-induced pain, p65 specific inhibitor pyrrolidinedithiocarbamate(PDTC) or BAY-11-7082, LV-p65 sh RNA were used in the present study.3. Results:(1) Significant mechanical allodynia, thermal allodynia and weight-bearing differences started to be observed at 2 weeks after carcinoma cells injection. Radiological images revealed bone destruction caused by carcinoma cells, and hematoxylin-eosin staining pictures showed significant tumor cells growth in the bone marrow cavity of tibia.(2) Tibia bone-specific DRG neurons were labeled by fluorescent dye Di I, and the majority of the Di I-labeled neurons were located in L2-L5 DRG.(3) After 2 weeks of tumor cell injection, the expression of P2X3 R in the tibia associated DRGs was significantly increased. Intrathecal injection purinergic receptor inhibitor suramin or P2X3 R and P2X2/3R potent inhibitor A317491 could attenuate hyperalgesia in a dose and time-dependent fashion.(4) p65 and p-p65 expression of tibia associated DRGs in cancer-induced rats were greatly enhanced. Intraperitoneal injection of p65 inhibitor PDTC or BAY-11-7082 could attenuate mechanical allodynia in a dose and time-dependent fashion.(5) LV-p65 sh RNA reduced the tibia specific neuronal excitability and alleviated pain behaviors of bone cancer rats.4. Summary These results indicate that P2X3 R and p65 were involved in the development of hyperalgesia induced cancer cells and the function P2X3 R was regulated by p65.Part two:Molecular and epigenetic mechanism of NF-κB participated in the regulation of P2X3 R expression1. Specific Aims: to clarify how p65 is involved in the regulation of P2X3 R expression, and to explore its mechanism of epigenetic regulation.(1) Correlation analysis was used to detect the correlativity between P2X3 R and p65 expression.(2) To investigate whether p65 and P2X3 are co-expressed in the tibial specific DRG neurons.(3) In order to verify whether p65 is involved in the regulation of P2X3 R expression, intraperitoneal injection of p65 inhibitor PDTC was used to detect whether the high expression of P2X3 R was reversed.(4) Intrathecal injection of p65 sh RNA lentivirus was to detect whether the expression of p65 and P2X3 R were inhibited.(5) Online software was used to predict the p65 binding sites on the Cp G islands of p2x3 r gene promoter, and to further verify the presence of p65 binding sites in Cp G islands by chromatin immunoprecipitation assay(Ch IP).(6) To detect the methylation status of Cp G island in the promoter of p2x3 r gene, and to investigate whether the combination of p65 and the promoter of p2x3 r gene enhanced.(7) To explore the mechanism of demethylation of p2x3 r gene promoter, namely further detection of the DNMT3 a and DNMT3 b expression, and active demethylation related protein Gadd45 a, MBD2, MBD4 and TDG expression.2. Methods:(1) Immunofluorescence staining method was used to detect the colocation of p65 and P2X3 R on the tibial specific DRG neurons;(2) The p65 binding sites in the Cp G island of p2x3 r gene promoter were predicted by online software Gene regulation, and was further analyzed with chromatin immunoprecipitation assays(Ch IP).(3) The status of DNA methylation on the Cp G island of p2x3 r gene promoter was determined by methylation specific PCR(MSP) and bisulfite sequencing PCR(BSP).(4) Expression of DNA methyltransferase(Dnmt3a, Dnmt3b), DNA damage-inducible protein alpha(Gadd45α), methyl Cp G binding domain protein MBDs(MBD2, MBD4) and DNA repair enzyme thymine DNA glycosylase(TDG) were measured with real-time PCR analysis.3. Results:(1) A correlation between p65 and P2X3 R expression in the tibia associated DRG of rat model with cancer-induced pain, and p65 and P2X3 R were co-localized in the tibia specific DRG neurons;(2) Intrathecal injection of p65 sh RNA lentivirus significantly inhibited the expression of p65 and P2X3 R, and can reduce the neuronal excitability and attenuate cancer-induced pain behavior.(3) There were 5 binding sites of p65 in the Cp G island of p2x3 r gene promoter by online software prediction. The chromatin immunoprecipitation assay confirmed that there were three p65 binding sites, a significant increase in binding activity of p65 with the first binding site of p2x3 r gene promoter in CIP rats compared to age-matched CON rats.(4) The methylation of Cp G island of p2x3 r gene promoter was significantly demethylated in bone cancer rats.(5) DNMT3 a and DNMT3 b m RNA expression of tibia specific DRGs in cancer-induced rats were greatly decreased. The expression of Gadd45α, MBD2, MBD4 and TDG was not significantly altered.4. Summary Tumor cell injection led to a significant demethylation of Cp G island in p2x3 r gene promoter and enhanced ability of p65 to bind the promoter of p2x3 r gene, which increased the expression of P2X3 R in rat tibia associated DRG. The demethylation of p2x3 r gene promoter may be mediated by the decreased expression of DNMT3 a and DNMT3 b. These results suggest that DNA methylation is involved in the occurrence and development of bone cancer pain.Conclusions:(1) Upregulation of P2X3 R expression is attributed to p2x3 r gene promoter DNA demethylation and p65 activation. The enhanced interaction of p65 and p2x3 r gene promoter would contribute to the enhanced expression of P2X3 Rs and the increased ATP currents, thus contributing to mechanical allodynia in bone cancer rats.(2) DNA demethylation is likely caused by decreased expression of DNMT3 a and DNMT3 b m RNA levels. Our data suggested for the first time that epigenetic mechanisms are involved in cancer pain.(3) P2X3 R and NF-κB p65 are involved in the formation of bone cancer-induced pain, indicating that P2X3 R and p65 might be potential targets for the treatment of cancer pain.