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目的采用茎环-逆转录实时荧光定量PCR方法,鉴定高脂饲料诱导的胰岛素抵抗小鼠肝脏组织中差异表达的mircoRNAs(miRNAs),即miR-1897-3p、miR-690和miR-7a-5p,并预测分析miRNAs调控的靶基因和功能,探讨胰岛素抵抗与差异表达的miRNAs的相关性。方法 30只雄性C57BL/6小鼠随机分为对照组和高脂饲料诱导的胰岛素抵抗模型组,设计茎环引物和实时荧光定量PCR特异引物,建立茎环-逆转录SYBR Green I实时荧光定量PCR方法,检测小鼠肝脏组织中miR-1897-3p、miR-690和miR-7a-5p的表达。统计学分析小鼠miR-1897-3p、miR-690和miR-7a-5p表达的差异。利用生物信息学软件预测miRNAs调控的靶基因,分析靶基因富集的基因功能(gene ontology,GO)和涉及到的信号转导通路及其靶基因蛋白的相互作用。结果经实时荧光定量PCR鉴定分析,与对照组相比,胰岛素抵抗组小鼠肝脏组织中miR-1897-3p、miR-690、miR-7a-5p表达下调(P<0.05)。生物信息学分析结果显示,有16种靶基因被两种差异表达的miRNAs调控,其中有8个靶基因可与4种以上的蛋白质相互作用,Rac1、Rhoa、Prkcz、Tgfbr2、Itch和Ube2d3蛋白位于网络的中心节点,且它们与胰岛素信号通路相关或存在交联。结论肝脏miR-1897-3p、miR-690和miR-7a-5p可能参与了胰岛素抵抗的病理生理过程,其机制可能通过调节靶基因的表达,影响了胰岛素信号通路的正常级联反应。
Objective To identify the differentially expressed mircoRNAs (miRNAs), namely miR-1897-3p, miR-690 and miR-7a-5p, in the liver tissue of insulin-resistant mice induced by high fat diet by using stem-loop-reverse transcription real- , And predict and analyze the target genes and functions regulated by miRNAs to explore the correlation between insulin resistance and differentially expressed miRNAs. Methods Thirty male C57BL / 6 mice were randomly divided into control group and high-fat diet-induced insulin resistance model group. The stem-loop primers and real-time PCR specific primers were designed and the stem-loop reverse transcription SYBR Green I real- Methods The expression of miR-1897-3p, miR-690 and miR-7a-5p in mouse liver tissue were detected. Statistical analysis of mouse miR-1897-3p, miR-690 and miR-7a-5p expression differences. Bioinformatics software was used to predict the target genes regulated by miRNAs, and the gene ontology (GO) was analyzed for its interaction with the involved signal transduction pathway and its target gene proteins. Results Real-time quantitative PCR analysis showed that compared with the control group, the expression of miR-1897-3p, miR-690 and miR-7a-5p in the liver of insulin resistance group was down-regulated (P <0.05). Bioinformatics analysis showed that 16 target genes were regulated by two differentially expressed miRNAs, of which 8 target genes could interact with more than 4 proteins. The Rac1, Rhoa, Prkcz, Tgfbr2, Itch and Ube2d3 proteins were located in Network’s central node, and they are associated with, or present with, the insulin signaling pathway. Conclusion Liver miR-1897-3p, miR-690 and miR-7a-5p may be involved in the pathophysiological process of insulin resistance. The mechanism may regulate the target gene expression and affect the normal cascade of insulin signaling pathway.