Supplementary Material for: Upstream stimulating factor 2 aggravates neuropathic pain induced in spinal nerve ligation-induced mice via regulating SNHG5/miR-181b-5p

Background: Upstream stimulating factor 2 (USF2) belongs to basic-Helix-Loop-Helix-Leucine Zipper transcription factor family, regulating expression of genes involved in immune response or energy metabolism network. Role of USF2 in neuropathic pain was evaluated. Methods: Mice were intraspinally injected with adenovirus for knockdown of USF2 (Ad-shUSF2), and then subjected to spinal nerve ligation (SNL) to induce neuropathic pain. Distribution and expression of USF2 was detected by western blot and immunofluorescence. Mechanical and thermal pain sensitivity were examined by paw withdrawal thresholds (PWT) and paw withdrawal latency (PWL). Chromatin immunoprecipitation (ChIP) and luciferase activity assays were performed to detect binding ability between USF2 and SNHG5. Results: The expression of USF2 was elevated and colocalized with astrocytes and microglia in L5 dorsal root ganglion (DRG) of SNL-induced mice. Injection of Ad-shUSF2 attenuated SNL-induced decrease of PWT and PWL in mice. Knockdown of USF2 increased level of IL-10, but decreased TNF-α, IL-1β, and IL-6 in SNL-induced mice. Silence of USF2 enhanced protein expression of CD206, while reduced expression of CD16 and CD32 in SNL-induced mice. USF2 bind to promoter of SNHG5, and weakened SNL-induced up-regulation of SNHG5. SNHG5 bind to miR-181b-5p, and miR-181b-5p to interact with CXCL5. Conclusion: Silence of USF2 ameliorated neuropathic pain, suppressed activation of M1 microglia and inhibited inflammation in SNL-induced mice through regulation of SNHG5/miR-181b-5p/CXCL5 axis. Therefore, USF2/SNHG5/miR-181b-5p/CXCL5 might be a promising target for neuropathic pain. However, the effect of USF2/SNHG5/miR-181b-5p/CXCL5 on neuropathic pain should also be investigated in further research.

研究背景：上游刺激因子2（Upstream stimulating factor 2, USF2）属于碱性螺旋-环-螺旋亮氨酸拉链（basic-Helix-Loop-Helix-Leucine Zipper）转录因子家族，可调控参与免疫应答或能量代谢网络的基因表达。本研究评估了USF2在神经病理性疼痛中的作用。实验方法：研究人员向小鼠脊髓内注射靶向敲低USF2的腺病毒（Ad-shUSF2），随后通过脊髓神经结扎（spinal nerve ligation, SNL）构建神经病理性疼痛模型。采用蛋白质印迹（western blot）与免疫荧光法检测USF2的分布与表达水平；通过缩爪阈值（paw withdrawal thresholds, PWT）和缩爪潜伏期（paw withdrawal latency, PWL）评估小鼠的机械性与热痛觉敏感性。采用染色质免疫沉淀（chromatin immunoprecipitation, ChIP）与荧光素酶活性实验，检测USF2与SNHG5的结合能力。实验结果：SNL造模小鼠L5背根神经节（dorsal root ganglion, DRG）中USF2的表达水平升高，且与星形胶质细胞、小胶质细胞存在共定位现象。注射Ad-shUSF2可缓解SNL诱导的小鼠缩爪阈值与缩爪潜伏期降低。敲低USF2可升高SNL造模小鼠体内IL-10的水平，同时降低TNF-α、IL-1β及IL-6的表达水平。沉默USF2可上调CD206的蛋白表达，同时下调CD16与CD32的表达水平。USF2可结合SNHG5的启动子区域，并削弱SNL诱导的SNHG5上调。SNHG5可靶向结合miR-181b-5p，而miR-181b-5p可与CXCL5发生相互作用。结论：沉默USF2可通过调控SNHG5/miR-181b-5p/CXCL5轴，改善SNL诱导小鼠的神经病理性疼痛，抑制M1型小胶质细胞活化与炎症反应。因此，USF2/SNHG5/miR-181b-5p/CXCL5轴有望成为神经病理性疼痛的潜在治疗靶点。不过，该信号轴对神经病理性疼痛的具体作用仍需在后续研究中进一步探究。