Cheng Shiqi, Zhang Yan, Chen Shuzhen, Zhou Yongliang
Department of Neurology, Jiangxi Provincial People's Hospital Affiliated to Nanchang University, No.92, Aiguo Road, Nanchang, 330006, Jiangxi Province, China.
Department of Neurosurgery, The Second Affiliated Hospital of Nanchang University, Jiangxi, China.
J Mol Neurosci. 2021 Jan;71(1):169-177. doi: 10.1007/s12031-020-01623-7. Epub 2020 Jun 29.
Traumatic brain injury (TBI) is one of the leading causes of death worldwide. Long non-coding RNAs (LncRNAs) have been reported to be closely associated with various diseases, but their roles in TBI has not been fully elucidated. The purpose of this study was to elucidate the underlying mechanism of LncRNA HOTAIR in TBI-induced microglial activation and inflammatory factor release. In vivo mouse TBI model and in vitro microglia activation model were established by Feeney's free-fall impact method and by LPS stimulation, respectively. The expression of LncRNA HOTAIR in activated microglia was detected by qRT-PCR. After shRNA knocked down, the expressions of LncRNA HOTAIR and microglia activation marker Iba-1 in microglia were detected by qRT-PCR and Western blot and by ELISA that detected the concentration of inflammatory factor in cell culture supernatants. The relationship between LncRNA HOTAIR and MYD88 in mouse microglia BV2 cells was observed by RNA pull-down assay. Furthermore, the effect of LncRNA HOTAIR on MYD88 stability was assessed by cycloheximide (CHX)-chase and by immunoprecipitation and ubiquitination assays that analyzed MYD88 ubiquitination. LncRNA HOTAIR was abnormally highly expressed in activated microglia. By Western blot and ELISA, the knockdown of LncRNA HOTAIR in microglia significantly repressed microglia activation and inflammatory factor release. By RNA pull-down assay, LncRNA HOTAIR could bind to MYD88 protein. Besides, by cycloheximide (CHX)-chase and immunoprecipitation and ubiquitination assays, the overexpression of the LncRNA HOTAIR enhanced the stability of MYD88 protein and inhibited Nrdp1-mediated ubiquitination of MYD88 protein. After the transfection of shRNA-HOTAIR and shRNA-HOTAIR+pcDNA-MYD88 into microglia, shRNA-HOTAIR could significantly inhibit the activation of microglia and the release of inflammatory factors, while these effects were reversed after the transfection of pcDNA-MYD88. Our experimental data indicated that LncRNA HOTAIR was highly expressed in activated microglia, and our further studies had found that the interference with LncRNA HOTAIR could repress microglia activation and inflammatory factor release via promoting Nrdp1-mediated ubiquitination of MYD88 protein.
创伤性脑损伤（TBI）是全球主要的死亡原因之一。据报道，长链非编码RNA（LncRNAs）与多种疾病密切相关，但其在TBI中的作用尚未完全阐明。本研究的目的是阐明LncRNA HOTAIR在TBI诱导的小胶质细胞激活和炎性因子释放中的潜在机制。分别采用Feeney自由落体撞击法和LPS刺激建立体内小鼠TBI模型和体外小胶质细胞激活模型。通过qRT-PCR检测激活的小胶质细胞中LncRNA HOTAIR的表达。shRNA敲低后，通过qRT-PCR、Western blot检测小胶质细胞中LncRNA HOTAIR和小胶质细胞激活标志物Iba-1的表达，并通过ELISA检测细胞培养上清液中炎性因子的浓度。通过RNA下拉试验观察小鼠小胶质细胞BV2细胞中LncRNA HOTAIR与MYD88之间的关系。此外，通过环己酰亚胺（CHX）追踪、免疫沉淀和泛素化试验评估LncRNA HOTAIR对MYD88稳定性的影响，这些试验分析了MYD88的泛素化。LncRNA HOTAIR在激活的小胶质细胞中异常高表达。通过Western blot和ELISA检测，小胶质细胞中LncRNA HOTAIR的敲低显著抑制了小胶质细胞的激活和炎性因子的释放。通过RNA下拉试验，LncRNA HOTAIR可以与MYD88蛋白结合。此外，通过环己酰亚胺（CHX）追踪、免疫沉淀和泛素化试验，LncRNA HOTAIR的过表达增强了MYD88蛋白的稳定性，并抑制了Nrdp1介导的MYD88蛋白泛素化。将shRNA-HOTAIR和shRNA-HOTAIR+pcDNA-MYD88转染到小胶质细胞后，shRNA-HOTAIR可显著抑制小胶质细胞的激活和炎性因子的释放，而在转染pcDNA-MYD88后，这些作用被逆转。我们的实验数据表明，LncRNA HOTAIR在激活的小胶质细胞中高表达，我们的进一步研究发现，干扰LncRNA HOTAIR可通过促进Nrdp1介导的MYD88蛋白泛素化来抑制小胶质细胞的激活和炎性因子的释放。
