新生未成熟大鼠缺氧缺血性脑损伤时微小RNA-200b对缺氧诱导因子1α的调控作用

The regulatory effects of microRNA-200b on hypoxia-inducible factor-1α in neonatal immature rats with hypoxic-ischemic brain damage

目的 探讨新生未成熟大鼠缺氧缺血性脑损伤(hypoxic ischemic brain damage,HIBD)时微小RNA 200b(micro RNA-200b,miR-200b)对缺氧诱导因子1α(hypoxia-inducible factors-1α,HIF-1α)的调控作用,为早产儿HIBD的治疗提供思路。 方法 选取3日龄新生Sprague-Dawley(SD)大鼠240只,随机分为单纯缺氧缺血组(HI组)、造模后双侧侧脑室注射miR-200b激动剂组、注射miR-200b拮抗剂组、注射miR-200b激动剂阴性对照剂组、注射miR-200b拮抗剂阴性对照剂组和正常对照组,每组40只。除正常对照组不予特殊处理外,其他各组均建立未成熟新生大鼠HIBD模型。应用实时定量聚合酶链反应检测各组大鼠侧脑室注射后12 h、1 d、3 d和7 d脑组织中HIF-1α的相对表达量,比较HIF-1α表达的变化。 结果 (1)与正常对照组比较,单纯HI组侧脑室注射生理盐水后12 h HIF-1α表达量开始升高,1 d达高峰,差异有统计学意义(P<0.05),随后逐渐下降,7 d与正常对照组接近,差异无统计学意义(P>0.05)。(2)单纯HI组与HI miR-200b激动剂阴性对照组及HI miR-200b拮抗剂阴性对照组HIF-1α表达量比较,差异无统计学意义(P>0.05),miR-200b纳米载体对HIF-1α表达无明显影响。(3)HI miR-200b拮抗剂组HIF-1α持续处于高表达状态,12 h明显高于单纯HI组,差异有统计学意义(P<0.05);注射后1、3、7 d HI miR-200b拮抗剂组与单纯HI组比较,差异无统计学意义(P>0.05)。HI miR-200b激动剂组HIF-1α表达持续低于单纯HI组,1 d时差异有统计学意义(P<0.05)。 结论 miR-200b过度表达抑制了HIF-1α表达,miR-200b低表达可上调HIF-1α表达水平,但作用时间有限。因此miR-200b有可能通过调控HIF-1α的表达参与新生大鼠HIBD后减轻脑损伤的调节。

Objective To study the regulatory effects of microRNA-200b (miR-200b) on hypoxia-inducible factors-1α (HIF-1α) in neonatal immature rats with hypoxic-ischemic brain damage(HIBD). Method A total of 240 three-day-old neonatal Sprague-Dawley (SD) rats were randomly assigned into six groups with 40 rats in each group: the hypoxic-ischemic group (HI group), intraventricular injection of miR-200b agomir, intraventricular injection of miR-200b antagomir, intraventricular injection of agomir negative control group, intraventricular injection of antagomir negative control group and the normal control group. The HIBD models of immature neonatal rats were established except for the normal control group. The relative expressions of HIF-1α in brain tissues of each group were detected using quantitative real-time-PCR at 12 h, 1 d, 3 d and 7 d after ventricular injection, and the changes of HIF-1α expression in each group were compared. Result (1) Compared with the control group, the expression of HIF-1α of the HI group began to increase 12 h after the injection of normal saline into the lateral ventricle (P<0.05), and reached the peak at 1d, with statistically significant difference (P<0.05), and then gradually decreased to the normal control group level at 7 d. (2) No significant differences of HIF-1α existed among the HI group and the HI+agomir negative control group and the HI+antagomir negative control group (P>0.05), and the miR-200b carrier had no significant effects on the expression of HIF-1α. (3) HIF-1α continued to be highly expressed after the injection of antagomir into the lateral ventricle of HI, and was significantly higher than the HI group at 12 h (P<0.05). No significant differences existed between the HI+antagomir group and the HI group at 1 d, 3 d and 7 d after antagomir injection (P>0.05). The expression of HIF-1α was constantly lower than the HI group after the injection of agomir, and significantly lower than the HI group at 1d after injection (P<0.05). Conclusion MiR-200b overexpression inhibits the expression of HIF-1α, and the low expression of miR-200b can increase the level of HIF-1α in a limited time window. Therefore, miR-200b may participate in the regulation of brain injury in neonatal rats after HIBD by regulating the expression of HIF-1α.