2023, Vol. 27 ›› Issue (2): 230-236

miR-132-3p targets and modulates Ptch1 to reduce neuropathic pain in mice with chronic constriction injury

Zhao Feng1, Fan Shaoqing1, Cheng Xiaoyan1, Li Xiaona1, Li Changsheng2, Ma Haojie1   

Abstract: BACKGROUND: The pathogenesis of neuropathic pain involving many pathological processes is complex. miR-132, which is abnormally expressed in neuropathic pain conduction pathway, affects the occurrence and development of pain.
OBJECTIVE: To investigate the role and mechanism of miR-132-3p in neuropathic pain in mice with chronic constriction injury.
METHODS: Mouse microglia BV2 cells were stimulated with 100 ng/mL lipopolysaccharide to establish an activation model. The expression of ionized calcium binding adapter molecule 1 (IBA1), a marker for microglia activation, was detected by western blot. The expression of miR-132-3p was detected by RT-qPCR. After transfecting miR-132-3p mimic or inhibitor into activated BV2 cells, the expression of IBA1 and P2X4 receptor (P2X4R) was detected by western blot. miR-132-3p targeted and regulated Ptch1, which was predicted and verified. pcDNA-Ptch1 or si-Ptch1 was transfected into BV2 cells for 24 hours and then stimulated with lipopolysaccharide for 24 hours. The expression of IBA1, P2X4R and Sonic Hedgehog (Shh) signaling pathway marker proteins was detected by Western blot. BV2 cells were co-transfected with miR-132-3p-mimic and pcDNA-Ptch1 for 24 hours and then stimulated by lipopolysaccharide for 24 hours. The expression of the above proteins was further detected by western blot. Thirty C57BL/6 mice were randomly divided into control group (n=6), sham group (n=6), model group (n=6), NC-mimic group (n=6) and miR-132-3p-mimic group (n=6). The latter two groups were injected intrathecally with NC-mimic and miR-132-3p-mimic, respectively. The mechanical withdrawal threshold and thermal withdrawal latency were monitored. The expression of miR-132-3p, Ptch1, P2X4R and Shh pathway marker proteins in dorsal root ganglion was detected by RT-qPCR and western blot to clarify the role and mechanism of miR-132-3p in chronic constriction injury-induced neuropathic pain.
RESULTS AND CONCLUSION: Compared with the control group, lipopolysaccharide stimulation promoted the activation of BV2 cells (P < 0.05) and down-regulated the expression of miR-132-3p (P < 0.05). miR-132-3p inhibited the activation of Shh signaling pathway through targeted regulation of Ptch1, and further inhibited the activation of BV2 cells and the expression of P2X4R. Overexpression of Ptch1 could reverse the effect of miR-132-3p-mimic (P < 0.05). The chronic constriction injury model was successfully established. Compared with the NC-mimic injection group, intrathecal injection of miR-132-3p-mimic could down-regulate the expression of Ptch1 (P < 0.05), inhibit the activation of the Shh signaling pathway (P < 0.05), reduce the expression of P2X4R (P < 0.05), and significantly increase the mechanical withdrawal threshold and thermal withdrawal latency of mice with chronic constriction injury (P < 0.01). Overall, miR-132-3p can reduce neuropathic pain in mice with chronic constriction injury by targeting Ptch1 and inhibiting the activation of Shh signaling pathway.
Key words: miR-132-3p, Ptch1, the Sonic Hedgehog (Shh) signaling pathway, chronic constriction injury, neuropathic pain, microglia