缺氧缺血性脑损伤新生大鼠松果体miRNA-182及Clock mRNA的表达变化

Expression profiles of miRNA-182 and Clock mRNA in the pineal gland of neonatal rats with hypoxic-ischemic brain damage

摘要:

目的 观察新生大鼠缺氧缺血性脑损伤(HIBD)后松果体内小RNA(miRNA)的差异表达,研究其在HIBD 导致的昼夜节律紊乱中的作用。方法 将7 日龄的新生Sprague-Dawley(SD)大鼠随机分为HIBD 模型组和假手术组,根据Rice-Vannucci 法制作HIBD 模型,24 h 后分别取两组松果体组织,通过miRNA芯片检测及实时荧光定量PCR 法(RT-PCR)筛选出HIBD 后高表达的miRNA,测定其在各组织(肺、肠、胃、肾、大脑皮层、松果体组织)中的表达差异。利用RT-PCR 技术分别测定两组在缺氧缺血后0、24、48、72 h松果体中高表达miRNA 及靶基因Clock mRNA 的表达变化。结果 miRNA 芯片结果结合RT-PCR 技术筛选出多个和HIBD 相关的miRNA,其中miRNA-182 表达差异明显。miRNA-182 在松果体组织中高丰度表达。HIBD后24 h、48 h miRNA-182 的表达水平高于对应时间点的假手术组(P<0.05);与对应时间点的假手术组相比,HIBD 后0 h Clock mRNA 表达水平升高,48 h 时降低,72 h 后明显升高(P<0.05)。结论 miRNA-182 可能参与了HIBD 后昼夜节律紊乱的病理生理过程。

Abstract:

Objective To study the changes of miRNA expression in the pineal gland of neonatal rats with hypoxic-ischemic brain damage (HIBD) and the possible roles of miRNA in the pathogenesis of circadian rhythm disturbance after HIBD. Methods Seven-day-old Sprague-Dawley (SD) rats were randomly divided into 2 groups: HIBD and sham-operated. HIBD was induced according to the Rice-Vannucci method. The pineal glands were obtained 24 hours after the HIBD event. The expression profiles of miRNAs were determined using GeneChip technigue and quantitative real-time PCR (RT-PCR). Then the miRNA which was highly expressed was selected. The expression levels of the chosen miRNA were detected in different tissues (lungs, intestines, stomach, kidneys, cerebral cortex, pineal gland). RT-PCR analysis was performed to measure the expression profiles of the chosen miRNA and the targeted gene Clock mRNA in the pineal gland at 0, 24, 48 and 72 hours after HIBD. Results miRNA-182 that met the criteria was selected by GeneChip and RT-PCR. miRNA-182 was highly expressed in the pineal gland. Compared with the shamoperated group, the expression of miRNA-182 was significantly up-regulated in the pineal gland at 24 and 48 hours after HIBD (P<0.05). Compared with the sham-operated group, Clock mRNA expression in the HIBD group increased at 0 hour after HIBD, decreased at 48 hours after HIBD and increased at 72 hours after HIBD (P<0.05). Conclusions miRNA-182 may be involved in the pathogenesis of circadian rhythm disturbance after HIBD.