摘要
目的:通过体外建立Hep G2细胞胰岛素抵抗模型,检测胰岛素抵抗状态下微小RNA-7-5p(miR-7-5p)及其预测靶基因Itch的差异表达,初步探讨miR-7-5p对Itch基因的靶向作用及其与胰岛素抵抗的关系。方法:采用适宜浓度的软脂酸诱导Hep G2细胞,建立体外胰岛素抵抗模型;基于生物信息学分析预测miR-7-5p可能作用的靶基因及其富集的相关信号通路;运用RT-q PCR和Western blot技术检测在胰岛素抵抗状态下miR-7-5p和Itch的表达变化。结果:0.25 mmol/L的软脂酸作用于Hep G2细胞24 h可诱导细胞产生胰岛素抵抗,RT-q PCR检测表明,与阴性对照组相比,胰岛素抵抗组的miR-7-5p表达下调(P<0.01)。生物信息学分析结果表明,miR-7-5p有相当数量的靶基因富集于泛素-蛋白酶体系统,其中E3泛素连接酶Itch基因是与胰岛素抵抗最为相关的靶基因;Western blot结果揭示,在胰岛素抵抗状态下,Hep G2细胞中Itch蛋白表达上调(P<0.01)。结论:miR-7-5p可能参与了胰岛素抵抗的病理生理过程,其机制可能通过靶向调控Itch基因的表达,进而直接或间接影响胰岛素信号通路的正常转导。
AIM： To preliminarily explore the relationship between microRNA-7-5 p（ miR-7-5 p） and Itch gene and their relationship with insulin resistance by establishing insulin resistance model of Hep G2 cells in vitro for detecting differential expression of miR-7-5 p and its predicted target gene Itch in the state of insulin resistance. METHODS： The insulin resistance model of Hep G2 cells was induced by suitable concentration of plamitic acid. The possible target genes and the associated signaling pathways of miR-7-5 p were predicted based on bioinformatic analysis. The expression levels of miR-7-5 p and Itch were detected by RT-q PCR and Western blot in the Hep G2 cells with insulin resistance. RESULTS：The Hep G2 cell model of insulin resistance was successfully induced by treatment with 0. 25 mmol/L palmitic acid for 24 h.Compared with negative control group,the expression level of miR-7-5 p detected by RT-q PCR in insulin resistance group was significantly decreased（ P〈0. 01）. Bioinformatic analysis showed that a considerable number of target genes of miR-7-5 p were enriched in the ubiquitin proteasome system. Among them,E3 ubiquitin ligase Itch gene was the most relevant target gene to insulin resistance. The results of Western blot showed that the protein expression of Itch was up-regulated in the Hep G2 cells under insulin resistance（ P〈0. 01）. CONCLUSION： miR-7-5 p may be involved in the pathophysiological process of insulin resistance,which may directly or indirectly affect the normal transduction of insulin signaling pathway by targeting Itch gene.
作者
吴亚柳
马玉
于曼丽
王文栋
郝敏
常晓彤
WU Ya-liu;MA Yu;YU Man-li;WANG Wen-dong;HAO Min;CHANG Xiao-tong(Key Laboratory of Clinical Diagnostics,Hebei North University,Zhangjiakou 075000,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2018年第7期1317-1322,1328,共7页
Chinese Journal of Pathophysiology
基金
河北省自然科学基金资助项目(No.C2011405015)
河北省高等学校科学技术研究重点项目(No.ZD2018076)
河北北方学院创新人才培育项目(No.CXRC1316)
