miR-146a对父代高脂高糖饮食背景下子代新生鼠肝脏胰岛素受体基因表达的影响  

Effects of miR-146a on hepatic INSR gene expression in F1 offspring of paternal rats with high-glucose-high-fat diet

作　　者：唐华 党伟[2] 魏颖莉 曹银利[4] Tang Hua;Dang Wei;Wei Yingli;Cao Yinli(Department of Gastroenterology, Second Affiliated Hospital of Air Force Military Medical University, Xi′an 710038, China;Xi′an Mental Health Center, Xi′an 710061, China;Zhejiang General Hospital of the Chinese People′s Armed Police Forces, Hangzhou 310004, China;Department of Neonatology, First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, China)

机构地区：[1]空军军医大学第二附属医院消化内科,西安710038 [2]陕西省西安市精神卫生中心,710061 [3]中国人民武装警察部队浙江总队医院,杭州310004 [4]河南省新乡医学院第一附属医院新生儿科,453100

出　　处：《中华内分泌代谢杂志》2019年第5期404-409,共6页Chinese Journal of Endocrinology and Metabolism

基　　金：陕西省重点研发计划(2017SF-244)。

摘　　要：目的探讨miR-146a对父代高脂高糖饮食(HGFD)背景下新生鼠肝组织胰岛素受体(insr)基因表达的影响及作用机制。方法取5周龄雄性SD大鼠随机分为:正常饮食组(ND组)及HGFD组,分组饲养3个月后与正常雌鼠交配。检测子代新生鼠血糖水平、葡萄糖耐量(GTT)及胰岛素耐量(ITT)。提取子代新生鼠肝组织miRNAs,合成cDNA。采用二代测序技术比较两组差异miRNAs,并通过实时荧光定量PCR(qPCR)的方法验证这些miRNA的表达差异。提取子代新生鼠肝组织基因组DNA、总RNA及总蛋白,qPCR比较父代HGFD对子代新生鼠肝组织中INSRmRNA相对表达量及INSR启动子甲基化水平的影响,分别分析INSR表达及甲基化水平与差异分子miR-146a表达水平的相关性。上调大鼠正常肝细胞系BRL-3A中miR-146a,观察miR-146a对肝细胞中INSR基因启动子甲基化水平(qPCR)及蛋白表达(Western-blot)的影响。结果HGFD组F1代雄性仔鼠葡萄糖耐量显著下降和胰岛素耐受。miRNA测序获得差异microRNAs60个,HGFD组上调的45个,下调的15个。qPCR检测提示其中8个miRNAs在扩大样本中仍有显著性差异,miR-146a在HGFD组仔鼠肝中表达上调超过10倍。miRNA-146a的表达水平与INSRmRNA的相对表达量负性相关,与INSR甲基化水平正相关。上调BRL-3A细胞中miR-146a表达,INSR基因启动子区的甲基化水平显著增强而蛋白表达则受到抑制。结论父代HGFD背景下F1代仔鼠肝组织中INSR表达抑制与miR-146a促INSR基因甲基化有关。Objective To study the effect of miR-146a on hepatic INSR gene expression in F1 offspring of paternal rats with high-glucose-high-fat diet (HGFD). Methods 5-week-old male SD rats were randomly divided into normal diet (ND) and HGFD groups. Male rats with ND or HGFD feeding for three months mated with normal female ones. Blood glucose concentration, glucose tolerance, and insulin tolerance in newborn male offspring rats were detected respectively. Differential miRNAs between ND and HGFD groups were compared using next generation sequencing and were then confirmed by real-time quantitative PCR (qPCR). The relative expression of INSR mRNA and the methylation level of INSR promoter in liver tissues of the offspring newborn rat were detected and the correlations between them and the relative expression of differential microRNA were analyzed respectively. In vitro, effects of miR-146a on expression and methylation of INSR gene in BRL-3A cells were detected using Western-blot assay and qPCR respectively. Results The fasting glucose concentration of different groups were without significant difference, but glucose tolerance and insulin tolerance of neonatal male rats in HGFD group were decreased significantly . Next generation sequencing has revealed 45 up-regulated miRNAs and 15 down-regulated miRNAs in HGFD group. Among them, differences of 8 miRNAs expression in the enlarged samples were confirmed by qPCR. miR-146a was up-regulated for more than 10 times in the liver of the offspring of HGFD group. Expression level of miRNA-146a was negatively correlated with the relative expression of INSR and positively correlated with the methylation level of INSR in livers of neonatal rats in HGFD group. In vitro, miR-146a (mimics) promoted the methylation of INSR gene and inhibited the expression of INSR in BRL-3A cells. Conclusion HGFD feeding to male SD rats leads to the inhibition of hepatic INSR gene expression in neonatal offspring via upregulating miR-146a.

关 键 词：microRNA-146a 甲基化 胰岛素受体基因 高脂高糖饮食 雄性 SD大鼠 

分 类 号：R587.1[医药卫生—内分泌]