miR221/222参与妊娠期糖尿病致子代异常的作用及机制研究

Gestational diabetes mellitus (GDM) is an important metabolic disease affecting pregnant women and fetal health. Epigenetic mechanism plays a key role in fetal adverse outcomes resulted from intrauterine hyperglycemia environment of women with GDM. Nevertheless,the exact mechanism is still unknown. In our previous study, we have screened differentially expressed microRNA in the serum speciemen of patients with GDM or pregnant mouse model, compared with their normal controls. Of note, miR221/222 was the most changed microRNA in GDM or during pregnancy. Moreover, we found that miR221/222 is ubiquitiously expressed and highly conserved among different species. By target gene predicting and validation experiment, we further demonstrated that miR221/222 overexpression inhibited the expression of cell proliferation-related genes, PTEN and P27. Taken together, miR221/222 expression is dramaticly changed during pregnancy and in the pathological process of GDM.It is most likely that miR221/222 deregulation in GDM influences fetal growth and metablism by regulating the expression of downstream target genes. In this project we intend to utilize clinical samples and GDM mouse model to explore the miR221/222 involved epigenetic mechanism underlying the impact of intrauterine hyperglycemia environment on the development and metabolic abnormalities in the offspring in vivo and in vitro. This study will provide new evidence for the molecular mechanism of intrauterine environment inducing offsrping diseases.

妊娠期糖尿病（GDM）是妊娠期重要的代谢性疾病，可造成不良妊娠结局并危及胎儿健康。表观遗传学机制在GDM宫内高糖环境致子代不良结局中扮演重要角色。我们前期工作中在妊娠小鼠模型及妊娠期糖尿病患者血清样本中筛选出差异表达的microRNA, 其中miR221/222变化最为显著，且miR221/222在全身各组织广泛表达。靶基因预测及验证实验发现，miR221/222过表达可抑制细胞增殖相关基因PTEN和P27表达。结果表明miR221/222是妊娠前后以及妊娠糖尿病病理过程中变化明显的高度保守性microRNA, 极有可能通过调控下游靶基因表达影响子代发育及代谢。本项目旨在前期重要发现的基础上，应用临床生物样本和妊娠期糖尿病小鼠模型，从体内、体外水平探讨妊娠期糖尿病宫内高糖环境影响子代生长发育的miR221/222表观遗传调控机制，为宫内不良环境所致子代疾病发生实现源头控制提供理论基础。

目的：胎盘娩出后妊娠期糖尿病（gestational diabetes mellitus, GDM）伴随的病理生理改变随即减退或消失。因此可以认定胎盘在GDM的病理改变中起着至关重要的作用。本研究通过探讨GDM胎盘中microRNA（miRNA）及其相应靶基因的差异表达，并同时探究miR-138-5p及其靶基因TBL1X在GDM发生发展中的作用，以期为GDM的发生机制提供理论依据。.方法：运用RNA-sequencing（RNA-seq）方法对8例GDM胎盘和8例正常组胎盘样本进行了miRNA及mRNA测序，获得GDM和对照组miRNA及mRNA表达谱。应用real-time PCR技术验证差异表达miRNA以及mRNA。应用划痕试验、transwell实验以及CCK-8检测细胞迁移以及增殖能力的改变。应用双荧光素报告酶实验验证TBL1X是miR-138-5p的靶基因。.结果:1）共筛选到281个差异miRNA以及32个差异mRNA，其中204个mRNA以及9个miRNA在GDM胎盘组织中上调，77个mRNA以及23个miRNAs在胎盘组织中下调。2）运用real-time PCR技术在20例GDM胎盘以及18例正常胎盘独立样本中进行验证，被选为验证的6个miRNA以及10个mRNA中，5 miRNAs以及8 mRNAs验证成功，包括5个上调的 mRNAs (TBL1X, NOTUM, FRMD4A, SLC16A2 and CLDN19) 以及 1个下调的 miRNA (miR-202-5p), 3个下调的 mRNAs (CCL18, HTRA1, SLC39A6) 和 4 个下调的 miRNAs (miR-138-5p, miR-210-5p, miR-3158-5p and miR-4732-3p)。3)通过对配对的miRNAs/mRNAs进行生物信息学分析，其功能主要涉及细胞发育及功能、器官形态等方面。3）miR-138-5p通过结合靶基因TBL1X改变滋养层细胞的迁移以及增殖功能。4）胎盘中miR-138-5p与TBL1X表达水平与母体血糖水平及胎盘重量相关。.结论：本研究首次获得了GDM胎盘组织中差异miRNA及mRNA表达谱。同时深入阐述了miR-138-5p及其靶基因TBL1X在GDM病理机制中的作用。