Analgesic Effect Of Down-Regulation For Spinal Protocadherin20by RNA Interference In Tibial Cancer Pain Rats

ObjectiveTo investigate the role of spinal Protocadherin20(PCDH20) in the rat model oftibial cancer pain.Methods1. The linearized vector GV118and prepared DNA sequence were constructed intotarget plasmid GV118-siRNA under the effects of T4DNA convertase, and the targetplasmids were transformed into competent cells. The grown colonies were identified bycolony PCR, and then the positive colonies were sequenced and aligned. Recombinantlentivector plasmids and the other two assisted plasmids were co-transfected into293Tcells by LipofectamineTM2000, and cell culture supernatant was collected after48h. Thevirus supernatant was concentrated and tittered in293T cells. The infection efficiency ofthe constructed virus was examined in neurons and the gene silencing effect was detectedby real-time quantitative PCR.2. Thirty-six SPF female SD rats,180to200g, were randomly divided into fourgroups with9rats in each group: Sham(S group), Tibial Bone Cancer Pain (BCP group), BCP+Control-LV (LC group) and BCP+PCDH20-LV (P group). The rat model of bonecancer pain was established by right tibia intramedullary injection of Walker256cells, andfor LC and P groups,4ul control-lentivirus and siRNA-PCDH20-lentivirus were injectedinto the ipsilateral spinal cord respectively on the same day. Mechanical WithdrawalThresholds (MWT) were measured by37400Dynamic Plantar Aesthesiometer at1thdbefore operation（T1）,3thd,7thd,14thd and21thd post-sarcoma inoculation（T2、T3、T4、T5）successively. Finally, rats were sacrificed at21thd post-sarcoma inoculation, and thedestruction of ipsilateral tibias in each group were observed with Hematoxylin and eosinstaining method(HE), while the expression of spinal PCDH20and postsynaptic density95(PSD95) were measured by realtime PCR, immunohistochemistry and western blot.Results1. siRNA–PCDH20-lentivirus was successfully synthesized, and the titer ofconcentrated virus was2×109TU/mL. The infection efficiency of the virus in neurons wasmore than72%when the MOI was10, and its’ gene silencing efficiency was about76.7%(P＜0.05) on the level of mRNA.2. Exception of Sham group, sarcoma implantation resulted in the significantdestruction of the tibia sclerotin and intramedullary infiltration of sarcoma cells in the othergroups. The expression of spinal PCDH20and PSD95significantly increased betweenBCP group and BCP+Control-LV group on the level of mRNA and protein (P＜0.05), andthe MWT in these two groups significantly decreased at the time of T3, T4and T5compared to the Sham group(P＜0.05). When compared to the BCP+Control-LV group,we found that the MWT of the BCP+PCDH20-LV group increased greatly at the time ofT4and T5, meanwhile, the expression of PCDH20and PSD95on the level of mRNA andprotein decreased (P＜0.05).Conclusion1. The siRNA-PCDH20-lentivirus was successfully constructed and the virus could effectively infect neurons and knock down the expression of PCDH20.2. The expression of spinal PCDH20and PSD95were increased significantly in the ratmodel of tibial cancer pain, and tibial cancer-induced mechanical allodynia was released bydown-regulating the expression of spinal PCDH20. The results of this research speculatedthat spinal PCDH20impacted the transmission ability of information in CNS by promotingthe generation of excitatory synapses, thus contributed to a new mechanism of tibial cancerpain.