The Mechanisms Of Relief Of Pain By Intrathecal Administration Of MiR-212 Antisense Locked Nucleic Acid In A Mouse Model Of Bone Cancer Pain

Objective:To observe the change of miR-212 in the development and maintenance of bone cancer pain and the mechanisms of the inhibitor of miR-212 on pain behaviors in mice of bone cancer pain.Methods:Part One:C3H/HeJ mice were randomly devided into group control and group tumor.2×105 osteolytic NCTC 2472 cells were inoculated into the intramedullary space of the right distal femur in group tumor to induce a mouse model of bone cancer pain. The mice of group control were inoculated by a-MEM without any cells. The pain behaviors, including paw mechanical withdrawal threshold and spontaneous lifting behaviors,were observed before inoculation and on 4 d,7 d,10 d, 14 d,21 d after inoculation. The spinal cord were taken after pain behaviors detection. Part Two:C3H/HeJ mice were randomly devided into group N, group S, group L, group L’ and group T. The mice of group L, group L’ and group T underwent surgery for bone cancer pain. On 14 d after inoculation, the mice of group L were intrathecal injected with LNA-anti-miR-212 (12 pmol/5 μl) while the mice of group L’were intratheal injected with LNA-negative control (12 pmol/5μl). The mice of group T and group S were intrathecal injected with RNase-free water (5μl). The pain behaviors were observed before inoculation and on 4 d,7 d,10d,14d,15d,16 d,17 d,18 d,19 d,20 d,21 d after inoculation. The lumar enlargement of mice were taken out to investigate the indexes using Western Blotting, real time PCR and immunofluorescence on day 21.Results:(1)Pain behaviors:There were no significant differences between the basal level of group control and group tumor. Compared with the basal level, the paw mechanical withdrawal threshold of both groups decreased while the spontaneous lifting behaviors increased on 4 d after inoculation (P< 0.05). The indexes of pain behaviors of group control recovered to basal level since 7 d after inoculation, while the indexes of the pain behaviors had significant differences in group tumor compared with that of basal level and group control. Since 19 d after inoculation, the significant increase of PMWT and decrease of spontaneous lifting behaviors were observed in group L compared with that of group T, while there was no effect of group L(2)real time PCR:Compare with the basal level, significant increase of miR-212 was observed in group control and group tumor on 4 d after inoculation. Since 7 d to 21 d after inoculation, the expression of miR-212 in group control recovered to the basal level while the expression of miR-212 in group tumor stayed at high level. On 21 d after inoculation, compared with that of group T, there was a significant decrease of the expression of miR-212 in group L, while there was no effect of group L’(3)Western Blotting:Compare with the basal level, significant increase of p-CREB was observed in group control and group tumor on 4 d after inoculation. Since 7d to 21 d after inoculation, the expression of p-CREB in group control recovered to the basal level while the expression of miR-212 in group tumor stayed at high level. On 21 d after inoculation, compared with that of group T, there was a significant decrease of the expression of p-CREB in group L, while there was no effect of group L’.There were no differences of CREB among all groups. Compared with that of group T, there was a significant decrease of the expression of BDNF in group L, while there was no effect of group L’(4) Immunofluorescence:Compared with that of group T, there was a significant decrease of the activiation of astrocytes in group L.Conclusion:miR-212 plays a important role in the development and maintenance of bone cancer pain. Intrathecal administration of LNA-anti-miR-212 could attenuate pain behaviors in a mouse model of bone cancer pain through downregulating the expression of p-CREB and BDNF as well as inactivating astrocytes in spinal cord.