Song Haipeng, Xu Na, Jin Shan
Department of Neurosurgery, Jinan First People's Hospital, Ji'nan, Shandong 250012, P.R. China.
Department of Emergency, Jinan First People's Hospital, Ji'nan, Shandong 250012, P.R. China.
Exp Ther Med. 2022 Jun 7;24(2):492. doi: 10.3892/etm.2022.11419. eCollection 2022 Aug.
microRNAs (miRNAs or miRs) have been reported to regulate the pathology of intracerebral hemorrhage (ICH). Therefore, the present study aimed to investigate the function of miR-30e-5p in rats with ICH with specific focus on Toll-like receptor (TLR)4. In the present study, collagenase type IV was used for the establishment of the ICH model in rats, prior to which the rats were injected with miR-30e-5p mimic or miR-30e-5p mimic + pcDNA3.1-TLR4 plasmid. The expression levels of miR-30e-5p and TLR4 were then measured using reverse transcription-quantitative PCR and western blotting. The potential interaction between miR-30e-5p and TLR4 was tested using the MicroRNA Target Prediction Database and dual-luciferase reporter and RNA immunoprecipitation assay. In addition, the concentration of TNF-α, IL-6 and IL-1β was measured using ELISA. The protein expression levels of TLR4/myeloid differentiation factor 88 (MyD88)/TIR-domain-containing adapter-inducing interferon-β (TRIF) signaling-associated molecules were measured by western blotting. Following induction of ICH, miR-30e-5p expression was downregulated, while TLR4 expression was upregulated. By contrast, injection with miR-30e-5p mimic rescued neuronal function while suppressing neuronal inflammation in rats following ICH; these effects were reversed by co-overexpression of TLR4. Furthermore, overexpression of miR-30e-5p inactivated TLR4/MyD88/TRIF signaling in rats with ICH; this was also reversed by overexpression of TLR4. Taken together, these results suggested that overexpression of miR-30e-5p exerted a protective role against neuronal deficit and inflammation caused by ICH in rats by targeting TLR4 and inactivating TLR4/MyD88/TRIF signaling.
据报道,微小RNA(miRNA或miR)可调节脑出血(ICH)的病理过程。因此,本研究旨在探讨miR-30e-5p在ICH大鼠中的作用,特别关注Toll样受体(TLR)4。在本研究中,使用IV型胶原酶建立大鼠ICH模型,在此之前,给大鼠注射miR-30e-5p模拟物或miR-30e-5p模拟物+pcDNA3.1-TLR4质粒。然后使用逆转录定量PCR和蛋白质印迹法测量miR-30e-5p和TLR4的表达水平。使用MicroRNA靶标预测数据库、双荧光素酶报告基因和RNA免疫沉淀试验检测miR-30e-5p与TLR4之间的潜在相互作用。此外,使用酶联免疫吸附测定法测量肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)的浓度。通过蛋白质印迹法测量TLR4/髓样分化因子88(MyD88)/含TIR结构域的接头诱导干扰素-β(TRIF)信号相关分子的蛋白质表达水平。ICH诱导后,miR-30e-5p表达下调,而TLR4表达上调。相比之下,注射miR-30e-5p模拟物可挽救ICH大鼠的神经元功能,同时抑制神经元炎症;TLR4的共过表达可逆转这些作用。此外,miR-30e-5p的过表达使ICH大鼠的TLR4/MyD88/TRIF信号失活;TLR4的过表达也可逆转这一现象。综上所述,这些结果表明,miR-30e-5p的过表达通过靶向TLR4并使TLR4/MyD88/TRIF信号失活,对ICH大鼠的神经元缺陷和炎症发挥保护作用。