Experimental Study Of Bone Cancer Pain Theraphy By RNA Interference Of P2X₃ Receptor MRNA

P2X₃ receptor,a subtype of ATP-gated ion channel,is selectively expressed in sensory neurons and plays a critical role in mediating chronic pain by transmitting harmful information.Therefore,chronic pain treatment by blocking P2X₃ receptors,will become an effective and hopeful therapeutic method without side effects.This study was focused on observing the therapy effects of blocking P2X₃ receptor expression in rat bone cancer pain model by RNA interference and exploring the underlying mechanism of this treatment.Hopefully,all these data can provide hints for gene therapy of chornic pain targeted on P2X₃ receptor in the future.Partâ… Changes of P2X₃ receptor expression and currents in a rat bone cancer pain modelObjective:To investigate the relationship of rat behavior and P2X₃ receptor expression in the rat bone cancer pain models induced by Walker 256 cells.Methods:â‘ Eighteen female SD rats were divided into two groups,each has nine.In bone cancer pain group,5ul(10⁴/ul)walker256 mammary gland carcinoma cells were transplanted into left tibia of rats by intra-tibia injection.In control group,5ul normal salines were injected into tibia of rats.The changes of allodynia and thermal hyperalgesia were observed at stages of 5,10,14 and 21 days after injection by Von-Frey filaments and CO₂ laser.Metastasises were observed in various organs.HE staining pathologic slices of bilateral tibia,lung were applied to evaluate tumor growth and metastasis under microscope. Successful rat model was set up when allodynia and thermal hyperalgesia was induced as well as the tumor growth was demonstrated by pathologic slices.â‘¡Twenty-one days after tumor cells intra-inoculation,rats were sacrificed by decapitation,and the L4-6 dorsal root ganglias were dissected out and put on ice for total RNA extraction.Changes of P2X₃ gene mRNA transcription and protein level were determined by RT-PCR and Immunoblot.â‘¢ATP induced currents usingα,β-meATP spraying were recorded by whole-cell patch clamp on acute dissipated DRG cells.Results:â‘ The threshold of mechanical allodynia and thermal hyperalgesia in left sides of bone cancer pain began to decrease 10 days after Walker 256 cells intra-tibia injection.The syndrome was progressively intensified.The mechanical allodynia and thermal hyperalgesia thresholds of right sides in cancer pain group rats and of both sides in control group rats were not changed during the observation period.Intra-tibial injections of walker256 cells produced a rapidly expanding tumor within the boundaries of the tibia, causing severe remodeling of the bone.â‘¡The mRNA level an protein level of P2X₃ gene were significantly higher in cancer pain group than that of the control group(P＜0.01).â‘¢The ATP induced currents were 1.44±0.23nA(n=5)in DRG cells derived from bone cancer pain rats,and were significantly higher than currents0.8±0.12nA(n=5)derived from control rats(P＜0.01).Conclusions:Rat bone cancer pain was successfully induced by Walker256 cells intra-tibia inoculation,and can be used to investigate mechanisms and therapy of bone cancer pain.Both of mRNA and protein level of P2X₃ receptor in bone cancer pain rats increase and induce P2X₃ currents.Therefore,these indicate that the P2X₃ dependent channel is potentially involved in the occurrence of bone cancer pain.Partâ…¡Constructing lentivirus expressing shRNA against P2X₃ receptorObjectives:To synthesis shRNA for blocking P2X₃ receptor expression in vitro and select the best lentiviral construct.Methods:â‘ Following siRNA design principles,three P2X₃ siRNAs,named 782,850,918,were synthesized and cloned into a vector(pSR-GFP vector)for siRNA expression.Full length P2X₃ cDNA was cloned in frame into pEGFP-N1,so the GFP-P2X₃ fusion protein could be expressed in mammalian cells,pEGFP-P2X₃ plasmids were co-transfected into 293T cells with P2X₃ RNAi vectors and the wild type vectors as control.Total cellular RNA was extracted at the time point of 36 hours post transfection,P2X₃ gene expression was analyzed by RT-PCR and Western-blot. The shRNA850 was shown to be the most efficient one and selected for the later work。②The shRNA850 construct was cloned into a lentiviral vector for siRNA expression.PRNAT-u6.2 was double digested with BamHâ… /Xhoâ… ,and ligated with DNA fragments encoding shRNA850.The ligation product was transformed into E.coli,recombinant plasmid was confirmed by sequencing.The recombinant vector was transfected into a lentiviral package cell line and the viral particles were obtained and purified.Viral titer was determined by real-time PCR.â‘¢Hippocampus neurons were infect by the lentiviral particles and the infection efficiency was evaluated.â‘£The packaged lentiviral particles were applied to infect 293T cell,and the expression of P2X₃ was accessed by Immunoblot. Results:â‘ Among three shRNAs,we saw the highest efficiency of No.850 blocking the expression of P2X₃ both at the level of mRNA and the protein vs pSR vector control(P＜0.01).â‘¡Lentiviral expression vector was constructed using shRNA850 and pRNAT-u6.2.The positive clone was applied to large scale viral preparation and the viral particles were purified,The titer was determined by real-time PCR and shown to be 5.3×10⁸Tu/ml.â‘¢The lentivirus particles effectively infected hippocampus neurons and its infection efficiency was nearly 100%.â‘£Expression of P2X₃ was blocked by the infection of recombinant lentiviral particles at MOI of 2,5,10 respectively.In control group,no significant difference of the GADPH expression was observed.Conclusions:Letiviral particles expressing P2X₃ shRNA could effectively infect neurons and block the expression of P2X₃.Therefore,this P2X₃ shRNA system is a powerful tool in our research wrok. Partâ…¢Effects of intrathecal injection of lentivirus expressingshRNA inhibiting P2X₃ gene on bone cancer pain ratObjective:The aim of the study was to observe the effects of P2X₃ gene silenced by intrathecal injection of letiviral expressed shRNA on mechanical allodynia and thermal hyperalgesia and the expression of P2X₃ receptors in dorsal root ganglia of bone cancar pain rats.Moreover,side effects of intrathecal injection of letiviral expressed shRNA were also observed.Methods:Thirty-one SD rats were randomly divided into five groups,in normal group and cancer pain group each has five,in therapy group and placebo group each has eight,another five normal rats were used to observe side effects by intrathecal injection of letiviral expressed shRNA.Normal rats were in normal group,bone cancer pain rats without any treatment were in cancer pain group.In cancer pain group and placebo group,10 days after induction of cancer pain and when obvious hyperalgesia was observed, 10ul letivirus particles(5.3×l0⁸Tu/ml)expressing shRNA850 or control shRNA were injected intrathecally into the rats separately.At various time points of 3d,7d, 2w,3 w,4 w,and 6 w after intrathecal injection,mechanical allodynia and thermal hyperalgesia values were determined.â‘¡Rats were sacrificed 6w after intrathecal injection and the L4-6 dorsal root ganglias were dissected on ice,P2X₃ receptor gene expressions in various groups were determined by RT-PCR and immunoblot separately.â‘¢Five normal rats receiving shRNA850 letivirus intrathecal injection were killed 6w after injection.Spine,DRG,lung,kidney,liver and heart were taken out and HE staining slices of these organs were making to obseve side effects of letivirus expressing shRNA850 after intrathecal injection.Results:â‘ The mechanical allodynia and thermal hyperalgesia values of therapy group were increased significantly 1 week after 10ul(5×10⁶Tu)letivirus particles containing shRNA850 were given compared with cancer pain group and placebo group(P＜0.01)at the same time point.Two weeks later,mechanical allodynia and thermal hyperalgesia values of therapy group showed no difference from that of the intact control group(P＞0.05)at the same time point.This therapeutic effect lasted 6w after intrathecal injection of letivirus particles containing shRNA850.â‘¡In concert with the findings,the expression of P2X₃ mRNA level and protein level were all decreased obviously in the therapy group compared with that of the cancer pain group and placebo control group(P＜0.01),while the expression of P2X₃ mRNA level and protein level in placebo group were same with the intact control group(P＞0.05).â‘¢We didn't see any side effects in normal rats after intrathecal injection of P2X₃ shRNAs expressing lentvirus throughout whole process.Conclusions:Under our observation,mechanical allodynia and thermal hyperalgesia of bone cancer pain can be alleviated for 6 weeks after intrathecal injection of P2X₃ shRNAs expressing lentvirus.Meanwhile we found this method can effectively silence the P2X₃ gene expression both at the protein and mRNA level.Moreover,we didn't see any side effects throughout whole process.