肝细胞内质网应激对tsRNA表达谱的影响及其功能研究

Expression and Function of tsRNA in Hepatocyte Endoplasmic Reticulum Stress

目的探讨肝细胞内质网应激相关的tRNA衍生性小RNA(tRNA derived small RNA,tsRNA)表达谱及其功能特征。方法将AML12细胞随机分为对照组和高脂组,采用棕榈酸刺激高脂组,建立肝细胞内质网应激模型。以RT-qPCR和Western blot法检测内质网应激标志蛋白GRP78和CHOP的表达,油红O染色法观察脂滴生成情况。通过高通量测序技术检测tsRNA表达谱并筛选出差异基因。利用生物信息学分析预测差异tsRNA的靶基因,并通过GO分析和KEGG分析研究差异tsRNA的潜在功能。结果与对照组比较,棕榈酸干预导致高脂组GRP78和CHOP表达在mRNA及蛋白水平均显著升高(P<0.05),脂肪变性显著加重。tsRNA测序得到38条差异tsRNA(倍数变化>2,P<0.05)。GO分析显示,差异tsRNA的靶基因主要调控类黄酮代谢、葡萄糖醛酸代谢和岩藻糖基化等功能。KEGG分析显示,差异tsRNA的靶基因在戊糖和葡萄糖醛酸酯的相互转化、淀粉和蔗糖代谢、cAMP及Rap1等肝细胞代谢相关的信号通路中高度富集。结论内质网应激可显著改变肝细胞的tsRNA表达谱,其功能可能与调控肝脏糖代谢及生物转化作用有关。

Objective To investigate the expression profile of tRNA derived small RNA(tsRNA)related to endoplasmic reticulum stress(ERS)and explore the functions of differentially expressed tsRNA.Methods The AML12 cells were randomized into control group and high-fat group.ERS model was established in the high-fat group with stimulation of palmitic acid.RT-qPCR and Western blot were used to detect the mRNA and protein expression level of GRP78 and CHOP,respectively.Oil red O staining was used to test the formation of lipid droplets.High-throughput sequencing was used to detect tsRNA expression profile and screen out the significantly changed tsRNAs.Bioinformatics analysis was used to predict the target genes of differential tsRNAs.Moreover,GO analysis and KEGG analysis were conducted to explore the function of differential tsRNAs.Results Compared with the control group,the mRNA and protein expression of GRP78 and CHOP were significantly increased in the high-fat group after treatment with palmitic acid.Hepatic steatosis was also significantly increased in the high-fat group.A total of 38 differential tsRNAs were filtered out by deep sequencing(fold change>2,P<0.05).GO analysis showed that target genes of tsRNA were mostly involved in flavonoid metabolic processes,glucoronate metabolic process and fucosylation.KEGG analysis showed that target genes of tsRNA were significantly enriched in signaling pathways associated with hepatocyte metabolism,such as pentose and glucoronate interconversions,starch and sucrose metabolism,Ras and Rap1 signaling pathway.Conclusion ERS exerts great impact on the expression of hepatocellular tsRNA profile.The differential tsRNAs may function to regulate the hepatic glucose metabolism and biotransformation.