The Analgesic Study Of PKCγ Down-Regulated By RNA Interference In Spinal Dorsal Horn In A Rat Model Of Bone Cancer Pain

Pain is one of the common symptoms in terminal cancer patients and the experienceof pain in people with cancer is highly variable and subjective because of severaldimensions,including of physiologic,sensory,affective,cognitive,behavioral,and socio-cultural,etc.Bone cancer pain has been described as a deep,boring sensation that achesand burns and accompanied by episodes of stabbing discomfort.Weight bearing oftenexacerbates this pain.In cancers associated with malignant invasion of the skeleton,metastatic bone destruction leads to increase morbidity and impaired quality of life as aresult of pain,fractures and other skeletal related events.So far,although there are opioid,diphosphonate,radiotherapy,chemotherapy and surgery for relieving cancer pain,it stillreported that a lot of cancer patients have inadequate and undermanaged pain controlbecause of the relative ineffectiveness and the side effects of current treatments.Therefore,it is very necessary for us to explore some novel mechanism-based approaches which havemore effective,long-lasting and less side-effect drugs to relieve the bone cancer pain formost patients. Spinal cord,especially spinal layerâ…¡,exerts important functions in the process oftransmitting and integrating nocuity information.It is very important to observe the pulseprojection and the changes of chemical substance in the spinal cord for clarifying themechanism about nocuity information transmission and regulation.Many study resultsmanifestate that PKCγin spinal dorsal horn not only participates nocuity informationtransmission and regulation,but also promotes the formation and development of spinalsensitization.During the development and maintenance of chronic pain,the expression ofPKCγevidently increases and PKCγprobably exerts important functions,so if theexpression quantity of PKCγcan be decreased effectively,perhaps the better analgesiceffect can be obtained.RNA interference technology (RNAi) is a post-transcriptional gene silencingtechnology (PTGS) which makes use of double short RNA(dsRNA) to specificallydegradate the target mRNA and to highly efficiently silence the corresponding geneexpression.RNAi considered as an effective gene knockout tool for studyingendogenous gene functions and signal pathways has been widespreadly applied toresearch in cell level and animal level,but the biggest obstacle about siRNA applicationis how to carry out the functions of siRNA lastingly and validly.Lentivirus vectors which are commonly used to transfering gene can infect non-mitotic phase cells and mitotic phase cells.Once lentivirus binds to cells,the gene oflentivirus can integrate to the genome of cells and become a part of stable constituent ofgenetic heredity which can pass down to filial generation during the cell fission process.Because the virulence gene has been rejected and the wild form of lentivirus is verydifficult,the lentivirus vector used to expressing siRNA can safely and lastinglydown-regulate the expression of gene in in-vitro and in-vivo experiments.Based on above theory,this study was designed four parts to evaluate the analgesiceffect when the expression of PKCγin the spinal dorsal horn was down- regulated bysiRNA interference. 1.After the rat models of cancer-induced bone pain (CIBP) were made,the changes ofPKCγexpression and the changes of nociceptive behaviors was observed,and thenwhether there has some relative relation was explored between the up-regulatedexpressiong of PKCγand the changes of nociceptive behaviors,including mechanicalwithdrawal threshold (MWT) and mechanical withdrawal duration (MWD).2.In the base of the first study,the CIBP models were intrathecally injected GF109203X(an inhibitor of PKCγ).When the PKCγfunction was inhibited,whether the MWT waslightened and the MWD was significantly prolonged or not were explored.And thenwhether PKCγdefinitly exerts important function in the mechanism of CIBP wasevaluated.3.After three lentivirus vectors of shRNA based on rat PKCγgene sequence wereconstructed and their interference effects were identified in vitro,a highly effectiveinterference target vector which would provide a basis to next experiment was selected.4.Based on the third result,the effective target lentivirus vector was injected into ratcavitas subarachnoidealis,the analgesic effect which would provide a theory andexperiment basis to chronic pain gene therapy and siRNA experiment in vivo wasevaluated. Methods and Results1:The expression and significance of PKCγin spinal dorsal horn in a ratmodel of cancer bone painMethods:Sixty-four female Wistar rats were selected and randomly divided into 3groups:Naive group (not received any surgery,n=16);Sham group (unilateral intra-tibialinjection of 10μl heat-killed cells,n=16) and CIBP group (unilateral intra-tibial injectionwith 10μl Walker 256 carcinoma cells,n=32).The changes of nociceptive behaviors(including MWT and MWD) were recorded on pre-operation and post-operation everyinterval 3 days.After the rats were sacrificed by perfusion with 4% paraform onpost-operation d6^th,d15^th and d21^st (4 rats every time point),the lumbar 4-6 spinal cord fordetecting the PKCγexpression changes through immunohistochemistry was removed andsectioned at 30μm on a freezing microtome.Meanwhile the western blot analysis methodwas also used to detect the PKCγexpression of CIBP group on pre-operation andpost-operation(d6^th,d15^th,d21^st).Results:The basic pain threshold has not any difference among all groups beforeoperation.After operation the MWT of CIBP group gradually decreased and the MWDevidently increased from day 6^th to day 21^st compared to that of na(?)ve group or sham group(P＜0.05).The immunohistochemistry result displayed that there has a little of PKCγexpression in L4～6 spinal dorsal horn in naive group.The average optical density (AOD) ofPKCγin CIBP group significantly up-regulated compared to sham group on d6^th,d15^th andd21^st (P＜0.05),meanwhile the average optical density(AOD) of PKCγin CIBP group ond15^th and d21^st was evidently more than that on d6^th (P＜0.05).The western blot result wasas same as the immunohistochemistry result.Correlation analysis suggested that there hasobvious positive correlation between the average optical density of PKCγin spinal dorsalhorn and the degree of pain. 2:Analgesic effect of intrathecal injection of PKCγinhibitor GF109203Xin a rat model of bone cancer painMethods:Thirty female Wistar rats were selected and randomly divided into 5 groupsas follows (n=6 each):Naive group (normal rats and not received any intervention);CIBPgroup (only unilateral intra-tibial injection with 10μl Walker 256 carcinoma cells);CIBP+Normal Saline (NS group,intrathecal injection of normal saline10μL to the CIBP rats);CIBP + DMSO group (DMSO group,intrathecal injection of DMSO 10μL to the CIBP rats);CIBP +GF109203X (GF109203X group,intrathecal injection of GF109203X 10μL to theCIBP rats).Changes of nociceptive behaviors were assessed by MWT and MWD beforeinjection of drugs as well as 15,30,45,60,75min after injection.After nociceptivebehavior assessments were finished the rats were sacrificed by perfusion with 4% paraform,the lumbar 4-6 spinal cord was removed and sectioned on a freezing microtome,and thenthe expression of PKCγwas detected through immunohistochemistry.Results:After intrathecal administration of GF109203X,the MWT of GF109203Xgroup was significantly higher than that of CIBP group (P＜0.05),and at 60 min afteradministration the MWT of GF109203X group was closed to the MWT of na(?)ve group(P＞0.05),meanwhile The MWD of GF109203X group evidently shorter than that ofCIBP group (P＜0.05).The MWT of DMSO group significantly increased and the MWDevidently decreased at 45,60,75min after administration compared to that of CIBP group(P＜0.05),but there had still significant differences in MWT and MWD between DMSOgroup and GF109203X group (P＜0.05).The expression levels of PKCγfailed to displaysignificant changes in spinal dorsal horn in the respective intrathecal injection group,including NS group,DMSO group and GF109203X group compared to CIBP group byimmunohistochemistry (P＞0.05). 3:Construction of shRNA lentivirus vector targeting rat PKCγgene andidentification of its interference efficiencyMethods:Three target sequences (19nt) were selected according to the rat PKCγ-mRNA sequence,whereafter the sense and antisense oligonucleotides were designed andsynthesized.After annealing,these double strand DNAs were cloned to the pLVTHMvector which contains H1 promoter and green fluorescent protein (GFP),meanwhile ageneral sequence was used as a negative control.All lentivirus particles produced by 293Tcell transfection with the vector plasmid,pMDLg-pRRE,pRsv-REV and pMD2G wereinfected the C6 cell and the Western blot was used to identify the inhibition efficiency ofthe constructed RNAi lentivirus vectors.Results:DNA sequencing demonstrated that the shRNA lentivirus vectors wereconstructed successfully.After infected C6 cells,the lentivirus of pLV-PKC₂,pLV-PKC₃could evidently decrease the expression of PKCγ,especially the inhibition efficiency ofpLV-PKC₂ (target site:+1913-+1931)) was the highest.4:Effect of analgesia by intrathecal injection ofPKCγ-shRNA lentivirus ina rat model of bone cancer painMethods:Twenty-four female Wistar rats which had been made CIBP model wererandomly divided into 4 groups as follows (n=6 each):CIBP group (not injection anydrugs);PBS group (intrathecal injection of PBS 10μL);pLV-Con group (intrathecalinjection of lentivirus pLV-Con 10μL);pLV-PKC₂ group (intrathecal injection of lentiviruspLV-PKC₂ 10μL).Changes of nociceptive behaviors including MWT and MWD wereassessed on pre-injection and post-injection every interval 3 days.After pain assessmentswere finished,the rats were sacrificed.The lumbar 4-6 spinal cord was removed anddetected the expression of PKCγthrough immunohistochemistry and Western blot. Results:After intrathecal administration,the MWT and MWD of PBS group andpLV-Con group had no any difference compared to CIBP group,but the MWT ofpLV-PKC₂ group significantly increased and the MWD evidently prolonged compared toCIBP group after 5 days in intrathecal injection of lentivirns pLV-PKC₂ (I.e.12^nd day aftermodels were made).The expression levels of PKCγfailed to display evident changes inspinal dorsal horn in PBS group and pLV-Con group compared to CIBP group byImmunohisto-chemistry and Western blot,however the PKCγexpression levels inpLV-PKC₂ group significantly reduced compared to CIBP group (P＜0.05).5:Statistical analysisAll of the analyses were performed by SPSS 11.0 software package.Measurementdata are presented as mean + standard deviation (SD).Group comparisons were made usinga One-way repeated measures ANOVA (behavioral testing data) and the other results usingANOVA.A value of P＜0.05 was considered statistically significant. Major Results:1.Through observing the pain behavioral changes of CIBP rats and the immunochemistrydetection,there has obvious positive correlation between the degree of pain and theexpression of PKCγin spinal dorsal horn.2.When the PKCγinhibitor GF109203X was intrathecally injected to CIBP rats,it couldbring about significant analgesic effect,which suggested that could exert importantfunction in the development/maintenance of bone cancer pain.3.The shRNA lentivirus vectors based on PKCγgene sequence were constructedsuccessfully and lentivirus pLV-PKC₂ could specifically,highly efficiently inhibitPKCγgene expression.4.When lentivirus pLV-PKC₂ was intrathecally injected to CIBP rats,it could generateevident analgesic effect and the expression of PKCγwas significantly down-regulated,which suggested that gene analgesic method mediated by siRNA could obtain definite,effective analgesic effect.Major Conclusion:In the development and maintenance of bone cancer pain,PKCγas an importantsignal molecular participates the transmission of nociceptive signal.After the lentiviruspLV-PKC₂ was intrathecally injected,it could bring about significant analgesic effect.Innovation:This study is a novel gene therapy based on the mechanism of CIBP.One side,itidentified signal molecular PKCγexerted important functions in the signal transmission ofCIBP;on the other hand,the lentivirus pLV-PKC₂ based on siRNA technology could produce significant analgesic effect,which suggested that gene analgesic therapy methodcould obtain definite,effective analgesic effect,and it could provide theory basis andexperiment basis to chronic pain therapy and the in vivo application of siRNA.