靶向胰岛素PI3K/Akt信号传导通路的环状RNAs与血糖异常转化的关联及作用机制研究

Dysregulation of insulin signaling pathway related gene expression is an early event in type 2 diabetes mellitus (T2DM). Recent studies found that circRNA may be involved in the regulation of gene expression by binding to miRNA as competitive endogenous RNA. To our knowledge, it is not clear whether circRNA can be used as biomarker of abnormal glucose transition. Our microarray analysis showed that there are 486 differential expressed circRNAs between the newly diagnosed T2DM patients and the normal controls. In this study, the candidate circRNAs targeting the insulin PI3K/Akt pathway will be selected by bioinformatics analysis, which could combine with T2DM specific miRNAs and their downstream target genes are the components of PI3K/Akt pathway. The potential roles of candidate circRNAs as biomarkers for abnormal glucose transition will be further evaluated in a functional community cohort. The support vector machine (SVM) predictive models will be established for T2DM and pre-diabetes. For the circRNAs which enter the predictive models, luciferase assays will be used to verify their direct effects on the corresponding miRNAs. The protein expression of important genes of the PI3K/Akt pathway will be observed directly under over-expression/ inhibition state of circRNAs. This study contributes revealing the mechanism of T2DM, identifying the individuals who are at high risk of T2DM or potential T2DM patients. In addition, this study will provide theoretical basis for finding a new generation of intervention targets for T2DM.

胰岛素信号传导通路相关基因表达失调是2型糖尿病（T2DM）发生的早期事件，而circRNA可能通过竞争性结合miRNA参与这些基因表达调控，但其能否作为血糖异常转化的生物标志物尚不清楚。前期芯片分析结果显示，新发T2DM患者与正常对照之间共486个circRNAs表达存在差异。本研究拟通过生物信息学分析筛选出能够结合T2DM特异性miRNA且下游靶基因为胰岛素PI3K/Akt通路组分的circRNAs；在功能社区人群队列中进一步明确其在血糖异常转化过程中的变化规律及意义，构建T2DM与糖尿病前期的支持向量机分类预测模型并进行验证；对于进入模型的circRNAs通过萤光素酶报告基因系统验证其对靶miRNA的结合作用，并在各circRNA过表达/抑制状态下观察其对PI3K/Akt通路关键基因蛋白表达的影响。为深入揭示T2DM发病机制，有效识别T2DM高危和早期人群，开发新一代干预靶点提供依据。

2型糖尿病（T2DM）已经成为影响公众健康的主要慢性病之一，及早发现T2DM高危人群和糖尿病前期人群并及时进行个体化干预，将成为T2DM防治的主要策略。相关基因表达失调是T2DM发生的早期事件，因而如果能够基于基因表达的改变来识别T2DM高危人群和早期人群对于T2DM的精准防治将具有重要意义。研究表明，circRNA可以作为miRNA海绵或ceRNA通过调节靶基因表达，参与疾病的发生发展，有望成为一种高效能的疾病诊断与预测标志物。前期研究结果显示，外周血单核细胞（PBMCs）中PI3K、Akt基因低表达，提示胰岛素信号通路受抑制。本研究基于新发T2DM患者与正常对照之间的circRNAs差异表达谱，通过生物信息学分析筛选出能够结合T2DM特异性miRNA且下游靶基因为胰岛素PI3K/Akt通路组分的circRNAs；在功能社区人群队列中进一步明确其在血糖异常转化过程中的变化规律及意义，构建T2DM与糖尿病前期的预测模型并进行外部验证；并通过萤光素酶报告基因以及RNA免疫共沉淀（RIP）实验进行生物学功能验证。结果显示外周血hsa_circ_0063425和hsa_circ_0056891低表达可以识别早期血糖异常且是胰岛素抵抗的预测因子，由2者组成的panel对于T2DM（AUC=0.837）以及空腹血糖受损（AUC=0.719）具有较高的识别能力，且在不同人群中具有良好的稳定性。荧光素酶实验和RIP实验进一步揭示hsa_circ_0063425通过与miR-19a-3p竞争性结合解除其对PI3K基因的抑制作用，同时hsa_circ_0056891通过充当miR-1-3p的分子海绵调控AKT，在外周血中三者之间表达水平仍存在相关关系。此外，还验证了潜在调控血糖转运体GLUT4的hsa_circ_0071336。本研究鉴定了T2DM的早期circRNA标志物，并且更加深入系统地认识了T2DM的发生病机制，为T2DM的精准防控提供了潜在新靶点。