The Effect Of WNK1 On Bone Cancer Rats And Its Possible Mechanism

Part one:Change in expression of WNK1 in spinal cordof a rat model of bone cancer painObjective To investigate the changes in the expression of WNK1 in spinal cord of a rat model of bone cancer pain.Methods 36 female SD rats,weighing 180-220g,were randomly divided into three groups:normal control group?group C?,sham operation group?group S?and bone cancer pain group?group BCP?.Group C was not given any treatment,and group S was injected into the bone marrow of left tibia with 5?l PBS solution while group BCP with 5?l WALKER 256 mammary gland cancer cell suspension(approximately 1×10⁵ cells).Mechanical paw withdrawal threshold?MWT?was measured at d1 before inoculation?baseline?and d3,6,9,10,11,12 after inoculation.Group S and C were sacrificed at d12while group BCP at d3,6,9,12 after inoculation and spinal cord?L4⁶?were removed at different time points for detection of WNK1 mRNA expression by qRT-PCR and WNK1protein expression by Western blot.Res?lts Compared with group C,group BCP's MWT started to decrease since d3?p<0.05?.The mRNA expression of WNK1 in spinal cord was up-regulated?p<0.05?from d3 showing an increasing trend over time until d9?peak?while the protein expression upregulated since d6 and also showed an increasing trend to d12?p<0.01?.Conclusion Expression of WNK1 in spinal cord of a rat model of bone cancer pain increased abnormally.Part two:The effect of intrathecal injection of WNK1 siRNA in a rat model of bone cancer painObjective To observe the change of MWT and the expression of KCC2 in spinal cord in a rat model of bone cancer pain after intrathecal injection of WNK1 small interfering RNA?siRNA?.Methods Three different siRNA duplexes targeting the rat WNK1were designed and synthesized,which were later transfected into PC12 neuronal cell line with lipofectamine2000.PC12 cells were divided into 6 groups?control,vehicle,mismatch,siRNA-3498,siRNA-5003 and siRNA-5461 group?.The expression of WNK1 was measured by qRT-PCR at 24h and Western blot at 48 hours after transfection in order to screen out the most efficient siRNA for knockdown of WNK1 in cell culture.In addition,30 female rats were randomly divided into 5 groups:sham operation?S group?,bone cancer pain group?BCP group?,siRNA-3498?BCP+intrathecal injection of WNK1siRNA?,mismatch?BCP+intrathecal injection of mismatch siRNA?and vehicle group?BCP+intrathecal injection of in vivo transfection reagent jetPEI?.The final concentration of siRNA as an siRNA–reagent complex was 3?g siRNA of 0.36?L jetPEI in 10?L 5%glucose solution.WNK1 siRNA,mismatch siRNA and jetPEI alone?defined as vehicle?were delivered to the lumbar region of the spinal cord via the intrathecal catheter on d9¹2,once a day.MWT was measured at d1 before inoculation?baseline?and d3,6,9,10,11,12after inoculation.The rats were sacrificed on d12 and spinal cord?L4⁶?were removed for detection of WNK1 and KCC2 mRNA expression by qRT-PCR and KCC2 protein expression by Western blot.Results Compared with the S group,the expression of WNK1 mRNA increased?p<0.01?and the expression of KCC2,both in mRNA and protein level,decreased?p<0.01?in BCP group.In addition,compared with BCP group,WNK1 mRNA was down-regulated?p<0.01?and KCC2,both in mRNA and protein level,were up-regulated?p<0.01?after intrathecal injection of WNK1 siRNA.However,there were no significant differences in the expression of WNK1 and KCC2 among BCP,vehicle and mismatch group?p>0.05?.Moreover,After 3 days of intrathecal injection of WNK1 siRNA,MWT of bone cancer rats was significantly increased?p<0.01?.Conclusion WNK1 may be involved in the rat model of bone cancer pain by inhibiting the expression of KCC2 in the spinal cord and intrathecal injection of WNK1siRNA can partially alleviate bone cancer pain.