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 m RNA in the pineal gland of neonatal rats with hypoxic-ischemic brain damage

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,24h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、72h松果体中高表达miRNA及靶基因Clockm RNA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24h、48hmiRNA-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0hClockm RNA表达水平升高,48h时降低,72h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。

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 Gene Chip 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 m RNA in the pineal gland at 0, 24, 48 and 72 hours after HIBD. Results miRNA-182 that met the criteria was selected by Gene Chip 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 m RNA 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.