miRNA-223抑制Pknox1基因调控糖尿病脂质代谢的分子机制研究

We found in earlier study, a significant down-regulation of miR-223 expression in non alcoholic fatty liver disease with type 2 diabetes mellitus compared with type 2 diabetes mellitus, and this change precede the visible histological changes and peripheral serum index. By miRNAs microarray analysis , we also found that in diabetic mellitus with non alcoholic fatty liver diease rats compared pure type 2 diabetes rats, miR-223 in plasma and liver tissue significantly decreased. This project aims to further study the regulation of miR-223 in blood glucose and insulin resistance as a functional mechanism in diabetes and non alcoholic fatty liver disease through animal models and in cell and molecular level , and predictive analysis combined with target genes of miRNA,we want to verify whether miR-223 through Pknox1 target genes regulating lipid metabolism which involving in the occurrence and development of non alcoholic fatty liver disease with type 2 diabetes mellitus, and expected the possibility of miR-223 as peripheral blood miRNA markers and early warning index.

我们前期发现， miR-223的表达在2型糖尿病伴非酒精性脂肪肝的患者中较单纯糖尿病患者血浆中明显下调，表达改变更先于肉眼可见的组织病理学变化及外周血清学指标，而且通过miRNAS微阵列分析亦发现糖尿病伴非酒精性脂肪肝的大鼠较单纯糖尿病大鼠中血浆和肝脏组织中miR-223的表达明显下降。本项目拟更深一步研究miR-223作为功能性的调节因子在糖尿病和高脂状态的病理生理中的作用机制，研究miR-223在人体内和动物模型中对糖尿病血糖水平和脂质代谢、胰岛素抵抗的调控作用，并在细胞和分子水平揭示其调控血糖和胰岛素抵抗的蛋白靶点，联合miRNA靶基因的预测分析，明确miR-223是否通过调控Pknox1靶基因编码的PREP1蛋白水平，影响脂质合成和分解的关键酶（SREBP1、FAS、ACC1、HMGR）调节脂质代谢，参与到2型糖尿病脂肪肝的发生发展，并探讨作为外周血可能的miRNA标记及预警新指标

2型糖尿病（T2DM）和非酒精性脂肪肝病(NAFLD)的共患形成恶性循环。本研究主要探讨T2DM 伴/或不伴NAFLD患者的miRNA谱表达变化及其作为预警指标可行性分析，并进一步研究差异表达的miR-17、miR-223等通过Pknox1对高糖环境下肝细胞及脂肪细胞脂质代谢的影响及可能机制。本研究采用STZ和高脂饮食建立T2DM合并NAFLD大鼠模型，应用miRNA芯片分析检测合并或不合并NAFLD的T2DM患者血浆miRNA表达前体，并用qRT-PCR进行验证，基于多元logistic回归分析的结果，建立了预测NAFLD存在的新指标。研究发现T2DM合并NAFLD患者血浆miR-17、miR-20a、miR-20b和miR-122水平明显高于非NAFLD患者，24.852×WHR-1.121×miR-122+1.988×LDL-21.838构建AUC是T2DM患者NAFLD的早期诊断和风险评估的潜在新工具。本研究进一步利用miRNA目标预测程序发现，Pknox1 mRNA序列的3′-UTR含量较高，是miR-17家族的保守靶位点。在链脲佐菌素和高脂饮食诱导的T2DM和NAFLD大鼠模型中，Pknox1肝组织表达增加与miR-17和miR-20a表达降低相一致。双荧光素酶报告分析进一步表明Pknox1是miR-17家族的有效靶基因。miR-17或miR-20a异位表达可显著抑制肝细胞Pknox1的表达,并通过改变胰岛素受体信号转导通路蛋白的磷酸化，降低细胞内甘油三酯和脂质的积累，显著提高HepG2和L02细胞的胰岛素敏感性和降低肝细胞脂肪变性。这些数据提示miRNA介导的Pknox1的调控策略可能为代谢性疾病提供新的治疗选择。