摘要
背景/目的：MicroRNA(miRNA)是一类新近发现的内源性非编码小RNA分子，在器官发育与分化、细胞增殖与凋亡、肿瘤形成、免疫反应等病理生理过程中发挥重要的调控作用。已有的研究表明，miRNA也参与了机体能量代谢平衡的调节，但miRNA与肝脏能量代谢相关的研究还很少。尽管“二次打击”学说等病理生理学机制可以部分解释非酒精性肝脏脂肪变性和脂肪性肝炎中肝脏损害逐渐进展的过程，但非酒精性脂肪性肝病发病和进展的很多具体机制尚不清楚。为了探索miRNA在非酒精性脂肪性肝病等肝脏异常代谢状态下的调控作用，本研究对非酒精性脂肪性肝病和1型糖尿病模型小鼠肝脏的miRNA表达谱进行了分析研究。
方法：采用miRNA芯片对10只非酒精性脂肪性肝病模型ob/ob小鼠，8只链脲霉素造模的1型糖尿病模型小鼠，以及8只正常对照C57BL/6小鼠的肝脏miRNA表达谱进行检测，并用微阵列显著性分析和聚类分析软件对芯片结果进行统计分析。miRNA芯片结果采用实时定量RT-PCR进行验证。本研究中发现的差异表达miRNA应用生物信息学miRNA靶基因预测算法软件进行靶基因预测分析。
结果：与正常对照小鼠相比，ob/ob小鼠发生脂肪变的肝脏miRNA中8个miRNA(miR-34a，miR-31，miR-103，miR-107，miR-194，miR-335-5p，miR-221，miR-200a)发生了显著上调，4个miRNA(miR-29c，miR-451，miR-2 1，miR-1 22)发生了显著下调；链脲霉素造模的1型糖尿病小鼠肝脏miRNA中，miR-34a发生了显著上调，miR-122发生了显著下调。比较ob/ob小鼠和链脲霉素造模的糖尿病小鼠肝脏miRNA表达谱，发现ob/ob小鼠肝组织中4个miRNA(miR-103，miR-31，miR-107，miR-126-3p)显著上调，2个miRNA(miR-100，miR-29c)显著下调。Ob/ob小鼠肝组织具有独特的miRNA表达谱形式，聚类分析可以将ob/ob小鼠肝脏样本与正常对照小鼠以及链脲霉素造模糖尿病小鼠的肝脏样本明确地区分开来。对本研究中发现差异表达miRNA的靶基因进行生物信息学预测，获得了一系列与非酒精性脂肪肝以及肝脏能量代谢相关的可能靶基因。
结论：本研究发现了一系列在非酒精性脂肪性肝病和1型糖尿病这两种肝脏异常能量代谢状态疾病模型中发生差异表达的miRNA，提示miRNA在肝脏能量代谢过程中发挥了重要的调控作用，并可能参与了非酒精性脂肪性肝病的病理生理学过程。扩展了我们对非酒精性脂肪性肝病生物学机制的认识，为今后可能的miRNA靶向治疗提供了线索。
Background/Aims:MicroRNAs (miRNAs) are a class of recently discovered,endogenous non-coding small RNAs,which play important roles in regulation of organdevelopment and differentiation,cell proliferation and apoptosis,carcinogenesis,immune response,etc.miRNAs are also demonstrated to be involved in regulation ofenergy metabolism,but only a few studies about the correlation between miRNA andhepatic energy metabolism have been reported.Although a widely accepted“two-hit”hypothesis may partially explain the progressive liver damage by non-alcoholicsteatosis and steatohepatitis,much of the pathogenesis of NAFLD remains undiscovered
Methods:The expression of microRNAs in livers of 10 ob/ob mice,8streptozotocin -induced type 1 diabetic mice and 8 normal C57BL/6 mice were analyzedby microRNA microarrays and analyzed by Significance Analysis of Microarrays.Theresults of miRNA microarrays were validated by quantitative RT-PCR.Putative targetsof the differentially-expressed miRNAs were predicted by bioinformatics algorithms formiRNA target prediction.
Results:Compared to normal C57BL/6 mice,ob/ob mice showed up-regulation of8 microRNAs (miR-34a,miR-31,miR-103,miR-107,miR-194,miR-335-5p,miR-221, and miR-200a) and down-regulation of 4 microRNAs (miR-29c,miR-451,miR-21,andmiR-122) in fatty livers.Up-regulation of miR-34a and down-regulation of miR- 122was found in livers of streptozotocin-induced diabetic mice.Comparison betweenmicroRNA expressions in livers of ob/ob mice and streptozotocin-administered micefurther revealed up-regulation of 4 microRNAs (miR-103,miR-31,miR-107,andmiR-126-3p) and down-regulation of 2 microRNAs (miR-100 and miR-29c) in livers ofob/ob mice.A distinctive microRNA expression pattern was identified in ob/ob mouseliver and hierarchical clustering of this pattern could clearly discriminate ob/ob micefrom either normal C57BL/6 mice or streptozotocin-administered mice.Bybioinformatics analysis of putative miRNA targets for the differentially-expressedmiRNAs,a list of target genes were predicted,most of which are relevant to the glucoseor lipid metabolism in liver and may play important roles in NAFLD
Conclusion:The present study demonstrates that distinct microRNAs are stronglydysregulated in non-alcoholic fatty liver disease and hyperglycemia,suggests animportant role of microRNAs in hepatic energy metabolism and implicates theparticipation of microRNAs in the pathophysiological processes of non-alcoholic fattyliver disease.These results broadened our biological understandings of NAFLD,andoffered useful clues for future clinical applications of miRNAs as potential targets.
引文
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