Abstract:

Objective To investigate the expression change of serum interleukin-1α (IL-1α) and interleukin-1β (IL-1β) concentrations and relative expression of microRNA (miRNA) in peripheral blood mononuclear cell (PBMC) of patients with acute severe traumatic brain injury (sTBI) and its molecular mechanism. Methods TargetScan Release 7.1 software was used to analyze miRNA regulating IL-1α and IL-1β genes. Enzyme-linked immunosorbent assay (ELISA) was used to detect expressions of serum IL-1α and IL-1β. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect miRNA relative expression of PBMC. Dual-Luciferase Reporter Assay System was constructed to verify the interaction between genes. Results IL-1α was the target gene regulated by miRNA-24-3p, and IL-1β was the target gene regulated by miRNA-383-3p. Compared with control group, the concentrations of serum IL-1α [(4.09 ± 2.32) ng/L vs. (0.56 ± 0.02) ng/L; t = 124.369, P = 0.030] and IL-1β [(3.99 ± 1.73) ng/L vs. (0.89 ± 0.03) ng/L; t = 163.123, P = 0.010] in sTBI group were significantly higher. In sTBI group, the relative expressions of miRNA-24-3p and miRNA-383-3p in PBMC were 23% and 17%, and the relative expressions of IL-1α and IL-1β were 390% and 420%. Dual-Luciferase Reporter Assay System showed the expressions of IL-1α (F = 40 154.000, P = 0.000) and IL-1β (F = 4015.000, P = 0.003) had significant difference in different groups. The expression of IL-1α in miRNA-24-3p group was significantly lower than that in control group (P = 0.000), miRNA-24-3p inhibitor group (P = 0.023), negative control group (P = 0.023) and negative inhibitor group (P = 0.023). The expression of IL-1β in miRNA-383-3p group was significantly lower than that in control group (P = 0.000), miRNA-383-3p inhibitor group (P = 0.000), negative control group (P = 0.000) and negative inhibitor group (P = 0.000). After transfected with clone IL-1α-mut-3'UTR and IL-1β-mut-3'UTR plamid, there were significant differences in different groups on expressions of IL-1α (F = 72.400, P = 0.001) and IL-1β (F = 37.000, P = 0.000). However, the expression of IL-1α in miRNA-24-3p group had no significant difference with control group, miRNA-24-3p inhibitor group, negative control group and negative inhibitor group (P > 0.05, for all), and the expression of IL-1β in miRNA-383-3p group had no significant difference with control group, miRNA-383-3p inhibitor group, negative control group and negative inhibitor group (P > 0.05, for all). Conclusions The concentrations of IL-1α and IL-1β in serum of sTBI patients are higher than that of normal controls. The mechanism may be that IL-1 α is negatively regulated by miRNA-24-3p and IL-1β is negatively regulated by miRNA-383-3p.

Key words: Craniocerebral trauma, MicroRNAs, Interleukin-1, Genes, Transfection, Reverse transcriptase polymerase chain reaction, Cells, cultured

摘要：

目的 探讨急性重型颅脑创伤患者血清白细胞介素-1α和 1β（IL-1α和 IL-1β）表达变化以及外周血单个核细胞微小 RNA（miRNA）相对表达量，并探讨其分子机制。方法 采用生物学信息软件TargetScan Release 7.1 分析调控IL-1α和IL-1β基因的 miRNA；酶联免疫吸附试验（ELISA）检测血清IL-1α和IL-1β表达变化，逆转录-聚合酶链反应检测外周血单个核细胞 miRNA 相对表达量；双荧光素酶报告基因载体验证基因之间相互作用。结果 IL-1α是 miRNA-24-3p 调控的靶基因，IL-1β是 miRNA-383-3p 调控的靶基因。急性重型颅脑创伤患者血清 IL-1α［（4.09 ± 2.32）ng/L 对（0.56 ± 0.02）ng/L；t = 124.369，P = 0.030］和 IL-1β［（3.99 ± 1.73）ng/L 对（0.89 ± 0.03）ng/L；t = 163.123，P = 0.010］水平高于正常对照者。急性重型颅脑创伤患者外周血 miRNA-24-3p 和 miRNA-383-3p 相对表达量为 23%和 17%，IL-1α和 IL-1β基因相对表达量为 390%和 420%。双荧光检测显示，各处理组细胞IL-1α基因（F = 40 154.000，P = 0.000）和IL-1β基因（F = 4015.000，P = 0.003）表达量差异有统计学意义，其中 miRNA-24-3p 组细胞 IL-1α基因表达量低于空白对照组（P = 0.000）、miRNA-24-3p抑制剂组（P = 0.023）、阴性对照组（P = 0.023）和阴性抑制剂组（P = 0.023），miRNA-383-3p 组细胞 IL-1β基因表达量低于空白对照组（P = 0.000）、miRNA-383-3p 抑制剂组（P = 0.000）、阴性对照组（P = 0.000）和阴性抑制剂组（P = 0.000）；经过转染克隆 IL-1α-mut-3'UTR 和IL-1β-mut-3'UTR 质粒后，各处理组细胞 IL-1α基因（F = 72.400，P = 0.001）和 IL-1β基因（F = 37.000，P = 0.000）表达量差异有统计学意义，但 miRNA-24-3p 组细胞IL-1α基因表达量与空白对照组、miRNA-24-3p抑制剂组、阴性对照组和阴性抑制剂组差异无统计学意义（均 P > 0.05），miRNA-383-3p组细胞IL-1β基因表达量与空白对照组、miRNA-383-3p 抑制剂组、阴性对照组和阴性抑制剂组差异无统计学意义（均 P > 0.05）。结论 急性重型颅脑创伤患者血清IL-1α和IL-1β水平高于正常对照者，其作用机制可能是IL-1α基因受 miRNA-24-3p负性调控、IL-1β基因受 miRNA-383-3p的负性调控。 

关键词: 颅脑损伤, 微 RNAs, 白细胞介素 1, 基因, 转染, 逆转录聚合酶链反应, 细胞, 培养的