The Role Of KIF17/mLin10 Interaction On Spinal NR2B Transport In The Maintenance Of Bone Cancer Pain In Mice

Objective:To explore the role of KIF17/mLin10 interaction on spinal NR2B transport pathway in the maintenance of bone cancer pain in mice.Methods:C3H/HeJ mice were randomly divided into normal group, sham group and tumor group.2×105 osteolytic NCTC 2472 cells were implanted into the intramedullary space of the right distal femur to induce mouse models of bone cancer pain. The sham group was inoculated by a-MEM without any cells. Normal group does no treatment. On the 14th day after inoculation, tumor group were further randomly divided into vehicle group, Myr-WP-13 group, Myr-RC-13 group. RC-13 group were treated by intrathecal injection of Myr-RC-13 10 μg (dissolved in 5μl 10% DMSO) one time per day for three days. Vehicle group and Myr-WP-13 group were treated by intrathecal injection of 5 μl vehicle (10% DMSO) or Myr-WP-13 10 μg at the same time. Mice were observed the changes of pain ethology indexes such as the paw withdrawal mechanical threshold and the spontaneous lifting behaviors before inoculation and at 3d,5d,7d, 10d,14d, and Id,3d,7d after administration. Lumbar intumescentia of mice in each group were taken out to investigate the expression level of NR2B, KIF17 and mLin10 western blotting, immunofluorescence, co- immunoprecipitation after pain behivor tests at the same time point.Results:(1) The paw withdrawal mechanical threshold of mice in sham group and tumor group decreased at day 3, but the difference between two groups tumor mice has no statistically significant. At day 5, the paw withdrawal mechanical threshold of these two groups recovered to the basic level. At 7 d after inoculation, quantification of the paw withdrawal mechanical threshold of tumor-bearing mice (0.90 ± 0.28) g decreased signigicantly (P< 0.05) as compared with baseline (1.93 ± 0.21) g and sham group (1.78 ± 0.31) g. Meanwhile, the spontaneous flinches of tumor group (4.43 ± 0.62) increased significantly (P< 0.05) as compared with baseline (2.25 ± 0.44) and sham group (11.11 ± 1.42). And the pain related behaviors gradually deteriorated over time and reached the peak at 14 days after operation. Consistent with the results of behavior tests, the expression of KIF17, NR2B and mLin10 proteins increased significantly compared with the baseline (P< 0.05) from day 3 or day 5 to day 14.(2) Co-immunoprecipitation:compared with vehicle group (0.80 ± 0.13), the ratio of mLin10/KIF17 interaction in mice treated with peptide Myr-RC-13 (0.23 ± 0.03) was siginificantly decreased (P< 0.05), while the isomer Myr-WP-13 had no effect.(3) The pain related behaviors of mice in RC-13 group alleviated obviously after repetitive administration. The paw withdrawal mechanical threshold increased and the number of spontaneous flinches decreased significantly of Myr-RC-13 group (P< 0.05). This analgesic effect reached the peak at day 1 after administration and recovered to the basic level at day 7. The results of western blot revealed that the expression of spinal KIF17, NR2B and mLin10 proteins of mice in Myr-RC-13 group was obviously decreased compared with vehicle group (P< 0.05). Furthermore, the intensity of immunofluorescence of spinal dorsal horn KIF17 and NR2B also decreased at day 1.Conclusion:KIF17/mLin10 interaction on spinal NR2B transport pathway may play an important role in the maintenance of bone cancer pain. Repetitive intratheal injection of Myr-RC-13, KIF17 competitive antagonist, can efficiently relieve pain related behaviors.