miRNA-21在孕期高脂暴露诱发子代肝内脂质过量沉积中的作用及机制研究

Prevalence rate of Nonalcoholic fatty liver disease (NAFLD) is gradually increased in recent years. It is characterized by accumulation of excessive lipids in the liver. Being exposed to maternal malnutrition during pregnancy increases the long-term risk of lipid metabolism disorder in the offspring liver, although the underlying mechanism is still unclear. In the prior cohort study, we found that maternal high triglyceride levels during pregnancy was associated with increased birthweight and higher lipids levels; Animal modal study showed that given high fatty diet after 8 weeks' old，HFD-F1 had more lipid stored in the liver than that of CD-F1, and continued lower expression of CPT1A，though similar expression of its key regulatory factors. miRNA Microarray followed with Targetscan database analysis explored the differential expressed miRNAs and predicted that miR-21 might increase accumulation of lipids in the liver by down regulated the expression of CPT1A. Therefore, we plan to prove the inter-action, lipids accumulation mechanism, and explore the epigenetic mechanism on the miR-21 regulation: 1) down/up express of mR-21, and prove the inter-action of the target gene downstream by firefly luciferase assay; 2) study the effect of miR-21 on the regulation of CPT1A by up/down miR-21 expression models; 3) Detect the DNA methylation level of miR-21 promotor region.

非酒精性脂肪肝病（NAFLD）患病率逐年升高，表现为肝内脂质过量堆积。宫内营养不良增加子代远期肝脂代谢紊乱风险，但潜在机制不明。我们前期队列研究证实母亲孕期高脂增加子代出生体重、血脂水平；动物研究发现同样成年后高脂饮食，宫内高脂子代（HFD-F1）较对照组（CD-F1）肝内脂质沉积增多，肝细胞CPT1A表达持续下调，而其主要调控因子表达不变，且CPT1A启动子区甲基化水平无改变。miRNA芯片筛选并预测miR-21可能通过下调CPT1A表达参与肝内脂质沉积。本项目拟在此基础上，通过体外细胞实验验证miR-21对CPT1A的调控作用；利用腺病毒转染构建miR-21过表达/敲除动物模型，进一步证实miR-21调控CPT1A引起远期脂质沉积；利用焦磷酸盐测序检测miR-21启动子区甲基化，探索宫内高脂引起子代肝内miR-21表达上调的表观遗传学机制，为胎儿源性成人疾病的发病机制提供理论依据。

成人慢性疾病，尤其是代谢性疾病的胎儿起源学说近年来受到广泛关注。母亲孕期宫内营养过剩，以高脂暴露、孕期过度增重为代表，对新生儿近期及成年远期代谢的影响成为研究热点之一。本项目聚焦亲代宫内营养过剩，以孕期高脂、高增重为重要暴露因素，与子代代谢紊乱，尤其是脂质代谢、体重、血压等的相关性，通过队列研究设计的流行病学调查发现母亲孕期持续的高甘油三酯水平（mTG）可增加妊娠高血压、先兆子痫、早产和子代高出生体重风险；控制体重并保持在标准孕期总增重的50th百分位点以下，可以降低高mTG导致的不良妊娠结局风险。大于胎龄儿（LGA）组在儿童期心血管代谢因子TC和LDL-c水平和TC/HDL-c比值显著高于对照组，结合宫内高脂暴露的LGA比例显著升高，提示宫内高脂环境可能是导致儿童期脂代谢紊乱的潜在因素。尽管已有潜在的证据支持胎儿宫内高脂暴露与远期非酒精性脂肪肝病的相关性，但是其中的机制仍不清楚。因此，我们对宫内高脂暴露子代和对照组子代的脐血全基因组DNA的甲基化进行了检测，针对差异基因进行IPA功能分析，获得一组与脂代谢紊乱的基因包括ALOX15，Apolipoprotein B gene (APOB), Carboxylesterase 1 gene (CES1),等，并扩大样本进行了飞行质谱验证。根据前期高脂动物模型实验，在实验组孕期给予60%高脂饲料与分娩后改为正常饮食。监测孕期母鼠的体重变化以及血脂水平，发现高脂组母亲孕期体重显著大于对照组；血脂TG、TC水平均显著高于对照组，并且实验组胎鼠的肝脏中的脂质显著增加，提示可能存在脂质代谢相关基因的重编程。我们通过PCR和WB检测比较了两组TG合成的上述关键酶，比较miR-21 和脂质代谢靶基因的蛋白表达，同时测定miR-21 启动子区甲基化，发现miR-21表达下降而TG合成酶表达显著增加，且miR-21区域甲基化水平部分CpG区域显著增加，提示甲基化的表观遗传学修饰可能参与了肝脏脂质代谢紊乱。本项目通过宫内高酯暴露的大鼠模型，从表观遗传学的角度阐述宫内高脂暴露引发的子代脂质代谢紊乱的可能机制，为代谢性疾病的宫内起源及表观遗传机制等开拓新思路，为阻断代谢性疾病的发生提供新的线索。

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