急性重型颅脑创伤患者外周血微小RNA和白细胞介素-1表达变化及分子机制研究

Expression and molecular mechanism of microRNA and interleukin-1 in peripheral blood of patients with acute severe traumatic brain injury

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。

Objective To investigate the expression change of serum interleukin-1α （IL-1α） and interleukin-1β （IL-1 β） 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-1 β. 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 1L-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-1 β-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 lL-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-1α is negatively regulated by miRNA-24-3p and IL-1β is negatively regulated by miRNA-383-3p.