microRNA-133参与追赶生长IUGR大鼠胰岛素敏感性表观遗传调控的分子机制研究

Intrauterine growth retardation especially that catch-up growth after birth (CG-IUGR) have been closely linked to metabolic syndrome in adulthood. However, the molecular mechanism is still unclear.Many studies indicated that epigenetics play key roles in uterine development and glycolipid metabolism, the current research focus and challenge are mode and degree of epigenetic inheritance in embryo development and catch-up growth. Applicants have found that key gene methylation and histone modification involved in development of metabolic disturbance in IUGR, however, the degree couldn't consist with metabolic and biochemical characteristic. Based on that, we consider that other epigenetic changes happened especially microRNAs as important post-transcriptional gene regulators,by which environmental factor could influence protein expression. microRNA-133 regulate target gene to contribute to differenciation of adipocyte and glucose transportation of skeletal muscle in IUGR. On the basis of our previous study,in this study, we investigate the expression alteration of miRNA-133 and MEF2.We also predict significant potential genes regulated by microRNA-133 through bioinformatics and validate them by luciferase reporter gene vector, in order to explore the regulated effect of microRNA on the glucose metabolism and insulin sensitivity in IUGR at different development stages. It will provide additional information on the mechanism of insulin resistance associated with IUGR, and provide a potential gene therapy approach to manage metabolic syndrome.

宫内发育迟缓尤其是生后出现追赶生长者(CG-IUGR)与成年期代谢综合征的发生高度相关，但具体机制尚不清楚。众多研究表明表观遗传在宫内发育及糖脂代谢中发挥重要作用，CG-IUGR表观遗传的改变成为当前研究重点和挑战。申请者前期研究发现DNA甲基化及组蛋白修饰参与CG-IUGR大鼠糖脂代谢异常发生发展过程，但甲基化程度改变与其代谢生化改变并不完全一致，据此我们推测非编码RNA的表观遗传调控也起关键作用。MicroRNA-133是调控脂肪细胞分化、骨骼肌发育和糖代谢的关键分子。在此基础上，本课题拟重点检测CG-IUGR大鼠各发育阶段microRNA-133及其靶分子的相互作用和信号通路分子PKB/Akt等程序化表达的改变，探讨microRNA-133调控CG-IUGR大鼠胰岛素敏感性的分子机制。本研究将加深宫内不良环境导致生长发育和代谢异常程序化改变的分子机制的的认识，并为干预治疗提供新靶点。

目的：宫内发育迟缓尤其是伴生后生长追赶者（CG-IUGR）与成年期代谢综合征紧密相关。前期研究表明表观遗传在宫内发育及糖脂代谢中起重要作用。本研究探讨microRNA-133在CG-IUGR伴糖代谢紊乱大鼠模型中的表达变化及调控作用。方法：通过孕期限食及生后减少哺乳数量构建CG-IUGR大鼠模型。4周龄时行葡萄糖耐量实验。构建microRNA-133a-sponge腺病毒和microRNA-133b-sponge腺病毒转染大鼠成肌细胞L6细胞系干扰microRNA-133功能。通过RT-PCR及Western Blotting 检测各组细胞系中及4周龄CG-IUGR大鼠及对照组骨骼肌组织中microRNA-133a和microRNA-133b的表达水平，及KLF15, Akt2, PGC-1α, GLUT4和 MEF2a的mRNA和蛋白表达水平。结果： IUGR大鼠2周龄时体重超过AGA组大鼠(p<0.05)，3周龄时鼻臀长超过AGA组大鼠(p<0.001)。葡萄糖耐量实验显示在30，60，120和180分钟时葡萄糖及胰岛素水平IUGR组较对照组明显增加。microRNA-133-sponge转染细胞组Akt2 和GLUT4 蛋白表达水平高于空白载体组。MicroRNA-133的表达在CG-IUGR组大鼠骨骼肌中显著降低，胰岛素通路关键分子KLF15, Akt2, PGC-1α, GLUT4和 MEF2a的 mRNA及蛋白水平在CG-IUGR组大鼠骨骼肌中显著降低。结论：本研究首次报道了microRNA-133 在CG-IUGR大鼠骨骼肌的表达变化。与在细胞系中对靶基因的负调控相反，在体内，靶基因与内源性microRNA-133同时降低。因此microRNA-133在CG-IUGR大鼠骨骼肌中的显著降低可能不是该模型机体内胰岛素通路受损的原因和刺激因素，反之，可能可缓解宫内不良不利影响需更进一步实验证实。