新的人LncRNA：lnc-GAT1调控妊娠期糖尿病患者脂肪组织慢性炎症反应的分子机制

Gestational diabetes mellitus (GDM) is defined as a glucose intolerance of varying severity with onset or first recognition during pregnancy. Insulin resistance is currently linked to the pathogenesis of GDM, although the molecular and cellular mechanisms involved in the development of GDM are not yet fully understood. Increasing evidence from clinical and experimental studies indicates that adipose tissue chronic inflammation, characterised by abnormal production of adipokines, is an essential factor linked to insulin resistance and GDM. Adipokines production is driven by a highly coordinated transcriptional cascade. Although some studies have identified several microRNAs involved in this process, molecular events regard to long noncoding RNAs (lncRNAs) during adipokines production remain to be explored. Using methods of High-throughput sequencing and Bioinformatics, we has identified a novel human lncRNA—lnc-GAT1, whose expression is down-regulated while its potential target gene TNFSF12 is up-regulated in abdomen subcutaneous adipose tissue of GDM women. Our preliminary data suggests lnc-GAT1 influences adipose tissue chronic inflammation through TNFSF12, which is known to be essential for inflammation. The present investigation aims to characterize and determine the physiological significance of the lnc-GAT1 with respect to adipose tissue chronic inflammation in GDM women. In order to investigate the novel role and potental mechanism of lnc-GAT1, the methods of RT-qPCR, western blotting, RNA pull down, MS, RIP and ChIRP will be also used in SGBS cell and primary human preadipocytes with overexpression/interferece lnc-GAT1 or/and TNFSF12. The results and conclusions will increase the understanding of mechanisms regulating insulin resistance and provide new insights into the pathophysiology of GDM. This knowledge also provides a framework for future investigations into the role of lncRNA in adipose tissue dysfunction and may ultimately help to identify novel therapeutic strategies aimed at ameliorating GDM.

脂肪组织慢性炎症反应是造成妊娠期糖尿病（GDM）胰岛素抵抗的关键因素之一。我们前期发现，新的人lnc-GAT1在GDM患者皮下脂肪组织中低表达，而其调节炎症反应的靶基因TNFSF12高表达，推测lnc-GAT1通过TNFSF12参与GDM脂肪组织慢性炎症反应。为阐明其分子机制，本项目拟采用GDM患者脂肪组织，利用RT-qPCR 、斑点杂交、FISH等技术观察lnc-GAT1的表达及定位情况，明确其生物学特性；采用lnc-GAT1和/或TNFSF12稳定高/低表达的SGBS细胞株和人原代前体脂肪细胞，通过检测细胞及培养液中炎性相关脂肪细胞因子的表达，分析lnc-GAT1的功能及其与TNFSF12的靶向调节关系；最后采用RNA pull down、MS、RIP等技术阐明lnc-GAT1调控TNFSF12基因表达的分子机制。结果将丰富lncRNA对GDM作用的认识，为GDM的防治提供新思路。

随着人们生活水平的提高和饮食结构的改变，妊娠期糖尿病(Gestational Diabetes Mellitus, GDM) 的发病率逐年升高。GDM 属高危妊娠，对母婴健康均具有深远影响。胰岛素抵抗是GDM的典型特征。尽管GDM胰岛素抵抗的分子机制不甚清楚，但大量研究表明，作为胰岛素靶器官之一的脂肪组织在GDM胰岛素抵抗的发生和发展中扮演着重要的角色。为了探究GDM发生和发展中脂肪组织的重要作用及潜在的诊疗靶点，本项目收集了GDM患者腹部皮下和大网膜的脂肪组织进行高通量全转录组测序，并利用自主研发的DLRAPom算法，筛选出1条功能未知的全新lncRNA，lnc-GAT1（GDM associated transcript-1）。随后我们利用人原代前体脂肪细胞为研究材料，利用 FISH和细胞核质分离技术，确定lnc-GAT1在细胞核和细胞质中皆有分布；利用RNAi和慢病毒过表达技术，证实lnc-GAT1可显著促进前体脂肪细胞成脂分化、增强成熟脂肪细胞的胰岛素敏感性、抑制炎性脂肪细胞因子的表达；随后利用DLRAPom算法、NRED、target scan软件预测及双荧光素酶报告基因证实，lnc-GAT1可与miR-96-5p和NR4A3的启动子区域靶向结合；在人原代脂肪细胞中敲低lnc-GAT1后miR-96-5p表达显著升高，而利用miR-96-5p inhibitor处理低表达lnc-GAT1的细胞，能显著逆转低表达lnc-GAT1对前体脂肪细胞分化和胰岛素敏感性的抑制作用。以上结果证实，lnc-GAT1通过ceRNA网络海绵结合miR-96-5p上调NR4A3表达，从而在GDM患者脂肪组织胰岛素敏感性和慢性炎症反应中发挥正调控作用。以上结果为进一步探究GDM患者胰岛素抵抗和脂肪组织慢性炎症的发病机制奠定理论基础；为改善GDM患者的脂肪组织功能提供新的诊疗思路。