【摘要】 目的:筛选高脂饮食诱导的胰岛素抵抗小鼠,以及应用Toll样受体4(Toll-like receptor4,TLR4)抑制剂TAK-242干预的胰岛素抵抗小鼠血浆差异表达的微小RNA(microRNA,miRNA),探讨胰岛素抵抗、TLR4和差异表达miRNAs的关系。方法:血浆标本来自前期实验的3组小鼠,即以普通基础饲料喂养(Low fat diet,LFD)的正常对照组、给予TAK-242的高脂饮食组(Treated high fat diet,HFD-T)和单纯高脂饮食组HFD-C,应用小鼠miRNA芯片检测血浆miRNA表达谱,筛选差异表达的miRNAs;采用实时荧光定量PCR方法验证芯片结果。应用生物信息学方法,以TLR4及其信号传导通路为中心,预测差异表达miRNAs的靶基因,并对靶基因分别进行GO和KEGG富集分析,以判定差异miRNAs主要影响的生物学功能或者通路。结果:miRNA基因芯片筛查结果显示,单纯高脂饮食组与正常饮食组比对,筛选出差异显著的miRNAs共计185种,其中6种miRNAs表达上调,179种miRANs表达下调;给予TAK-242的高脂饮食组与正常饮食组比对,筛选出差异显著的miRANs共计171种,均表达下调;给予TAK-242的高脂饮食组与单纯高脂饮食组比对,筛选出差异表达的miRNAs共计13种,均为下调。生物信息学分析结果显示,以TLR4为中心挖掘与其互作的蛋白质,共发现有10种蛋白;在TAK-242给予的高脂饮食组与单纯高脂饮食组中差异表达倍数均在1 000以上的4种miRNAs(mmu-miR-3095-3p、mmu-miR-5113、mmu-miR-709和mmumiR-335-3p),可在TLR4的互作蛋白质或Toll样受体信号通路中找到其作用的靶基因,发现这些靶基因74%属于生物过程基因,其中转录调控因子占82%。实时荧光定量PCR检测mmu-miR-3095-3p、mmu-miR-5113、mmu-miR-709和mmu-miR-335-3p水平,变化趋势与芯片结果一致。结论:胰岛素抵抗发生时,血浆miRNAs表达谱存在改变,此变化与TLR4及其信号通路相关。该研究结果丰富了胰岛素抵抗发生的机制,为寻找胰岛素抵抗血浆miRNAs诊断标志物提供了实验依据。更多还原

【Abstract】 Objective: To screen the plasma microRNAs( miRNAs) of differential expression in a high fat diet-induced insulin resistance in mouse models; further investigations on the mice with insulin resistance treated by TLR4 inhibitors TAK-242,and to study the changes of plasma miRNAs expression profile and the relationship among TLR4,miRNAs and high fat diet-induced insulin resistance. Methods: The plasma samples were from 3 mouse groups of previous study,namely,the control group with general basic diet( low fat diet,LFD),TLR4 inhibitors TAK-242 treatment group with a high fat diet( HFD-T) and the high fat diet control group( HFD-C). The differential expressed miRNAs was screened by expression profiling of plasma miRNAs,which was detected using mouse miRNA microarray. The quantitative Real-Time PCR( qRT-PCR) was used to verify the results of microarray. The target genes of differential expressed miRNAs were predicted in TLR4 signaling pathway using bioinformatics methods,and the GO and KEGG database molecular annotation system were used to investigate the main effects of the miRNAs targeted genes on the biological functions or signal pathway. Results: The screening results of miRNA microarray chip showed that,comparing miRNAs expression between HFD group and LFD control group,185 miRNAs were significant in the high fat diet group,including 6 up-regulated and 179 down-regulated miRANs. A significant difference of miRANs was also found between HFD-T group and LFD control group,the total number of differential expression miRNAs was 171,and all of them were down-regulated. Comparing miRNAs expression between HFD-C group and HFD-T group,13 miRNAs were significant in HFD-T group,all of them were down-regulated. Bioinformatics analysis results showed that a total of 10 interaction proteins with TLR4 were predicted; the difference of mmu-miR-3095-3p,mmu-miR-5113,mmu-miR-709 and mmu-miR-335-3p expression levels was more than 1 000 times between HFD-C group and HFD-T group,and their target genes can be found in TLR4-interaction protein or Toll like receptor signaling pathway; GO and KEGG analysis showed 74% of these target genes belonged to the biological processes genes,and the transcription factors accounted for 82%. The expression of mmu-miR-3095-3p,mmu-miR-5113,mmumiR-709 and mmu-miR-335-3p detected by qRT-PCR exhibited the similar patterns of down regulation to those shown in microarray results. Conclusion: When insulin resistance occurs,there is a change in plasma miRNAs expression profile,this change is associated with TLR4 and its signaling pathways. The finding enrichs the possible mechanisms of insulin resistance and provides a basis for finding miRNAs diagnostic markers for early diagnosis of insulin resistance.更多还原