Analgesic effect of lentivirus-mediated RNA interference of the expression of glial cell line-derived neurotrophic factor gene on bone cancer pain

Abstract

Objective To observe the analgesic effects of lentivirus-mediated RNA interference of the expression of glial cell line-derived neurotrophic factor (GDNF) on bone cancer pain.Methods (1)Lvs-siGDNF was constructed and bone cancer pain models were established by intra-tibial injection of MRMT-1 cells.(2) The rats were randomly divided into 4 groups according to different drugs which were infused into subarachnoid space through the intrathecal tubes:psiHIV-U6,psiHIV-GDNF-mU6,mor-phine,and normal saline.(3) The pain threshold at different time points (normal rats,7 days and 14 days after dosing) was measured.(4) The changes of pain threshold were observed from preoperative day to 7 days and 14 days after administration.The levels of GDNF,glial fibrillary acidic protein (GFAP),and Fos were detected by immunofluorescent staining technique and Western blotting.Results (1) As compared with control group (0.117 ±0.060) and transfection group (0.113 ±0.072),GDNF siRNA sequence 3 (0.045 ± 0.034) was the most effective interference segment confirmed by Q-PCR (P ＜ 0.05).The percentage of fluorescent cells to the brightfield was 0.87.TLvs-siGDNF was 3.48 × 108 pfu/ml.(2)Thermal pain threshold at 7th day (3.80 ±0.69) and 14th day (3.71 ± 1.10),and mechanical pain thresh-old at 14th day (2.58 ± 1.93) were all increased in siGDNF group than normal saline group (1.38 ± 1.01,1.11 ± 1.02,and 1.53 ±0.69) and psiHIV-U6 group (1.39 ± 1.18,0.67 ±0.27,and 1.21 ± 1.06)(P ＜ 0.05),while there was no significant difference in mechanical pain threshold at 7 day after administration (P ＞ 0.05).(3) RNA interference effectively decreased the expression level of GDNF in siGDNF group (P ＜0.05).(4) As compared with other groups,the expression levels of Fos and GFAP in the spinal dorsal horn were decreased in morphine group and siGDNF group (P ＜ 0.05).Conclusion The analgesic effect of siGDNF is similar to that of morphine in bone cancer rats,and siGDNF worked effectively more than 2 weeks.SiGDNF realized analgesic effect probably by inhibiting the astrocyte activation. Key words: Bone cancer pain; Glial cell line-derived neurotrophic factor; RNA interference; Lentiviral