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（LncRNA）与多种疾病密切相关，但它们在 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症因子释放中的潜在机制。分别采用Feeney的自由落体冲击法和LPS刺激建立体内小鼠TBI模型和体外小胶质细胞活化模型。qRT-PCR检测LncRNA HOTAIR在活化小胶质细胞中的表达。shRNA被击倒后，通过qRT-PCR和Western blot检测LncRNA HOTAIR和小胶质细胞活化标志物Iba-1的表达，ELISA检测细胞培养上清液中炎症因子的浓度。RNA pull-down法观察小鼠小胶质细胞BV2细胞中LncRNA HOTAIR与MYD88的关系。此外，LncRNA HOTAIR 对 MYD88 稳定性的影响通过放线菌酮 (CHX)-chase 以及分析 MYD88 泛素化的免疫沉淀和泛素化测定来评估。LncRNA HOTAIR 在活化的小胶质细胞中异常高表达。通过蛋白质印迹和 ELISA，小胶质细胞中 LncRNA HOTAIR 的敲低显着抑制了小胶质细胞的活化和炎症因子的释放。通过RNA pull-down分析，LncRNA HOTAIR可以与MYD88蛋白结合。除了，通过放线菌酮 (CHX)-chase 和免疫沉淀和泛素化测定，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。shRNA-HOTAIR和shRNA-HOTAIR+pcDNA-MYD88转染小胶质细胞后，shRNA-HOTAIR能显着抑制小胶质细胞的活化和炎症因子的释放，而pcDNA-MYD88转染后这些作用被逆转。我们的实验数据表明LncRNA HOTAIR在活化的小胶质细胞中高表达，我们进一步的研究发现干扰LncRNA HOTAIR可以通过促进Nrdp1介导的MYD88蛋白泛素化来抑制小胶质细胞的活化和炎症因子的释放。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。shRNA-HOTAIR和shRNA-HOTAIR+pcDNA-MYD88转染小胶质细胞后，shRNA-HOTAIR能显着抑制小胶质细胞的活化和炎症因子的释放，而pcDNA-MYD88转染后这些作用被逆转。我们的实验数据表明LncRNA HOTAIR在活化的小胶质细胞中高表达，我们进一步的研究发现干扰LncRNA HOTAIR可以通过促进Nrdp1介导的MYD88蛋白泛素化来抑制小胶质细胞的活化和炎症因子的释放。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。shRNA-HOTAIR和shRNA-HOTAIR+pcDNA-MYD88转染小胶质细胞后，shRNA-HOTAIR能显着抑制小胶质细胞的活化和炎症因子的释放，而pcDNA-MYD88转染后这些作用被逆转。我们的实验数据表明LncRNA HOTAIR在活化的小胶质细胞中高表达，我们进一步的研究发现干扰LncRNA HOTAIR可以通过促进Nrdp1介导的MYD88蛋白泛素化来抑制小胶质细胞的活化和炎症因子的释放。shRNA-HOTAIR和shRNA-HOTAIR+pcDNA-MYD88转染小胶质细胞后，shRNA-HOTAIR能显着抑制小胶质细胞的活化和炎症因子的释放，而pcDNA-MYD88转染后这些作用被逆转。我们的实验数据表明LncRNA HOTAIR在活化的小胶质细胞中高表达，我们进一步的研究发现干扰LncRNA HOTAIR可以通过促进Nrdp1介导的MYD88蛋白泛素化来抑制小胶质细胞的活化和炎症因子的释放。shRNA-HOTAIR和shRNA-HOTAIR+pcDNA-MYD88转染小胶质细胞后，shRNA-HOTAIR能显着抑制小胶质细胞的活化和炎症因子的释放，而pcDNA-MYD88转染后这些作用被逆转。我们的实验数据表明LncRNA HOTAIR在活化的小胶质细胞中高表达，我们进一步的研究发现干扰LncRNA HOTAIR可以通过促进Nrdp1介导的MYD88蛋白泛素化来抑制小胶质细胞的活化和炎症因子的释放。