邻苯二甲酸二丁酯暴露对大型溞miR-153及AKT基因表达的影响和功能分析

Expression and Function Analysis of miR-153 and AKTgene in Daphnia magna Exposed to Dibutyl Phthalate

microRNAs参与调控动物所有的生命过程,包括生长、发育、生物和非生物应激反应等。microRNAs与其调控的靶基因已被证实参与水生动物对环境胁迫的应答。为了探究miR-153的生物学功能及邻苯二甲酸二丁酯(DBP)暴露对大型溞miR-153及其靶基因AKT表达的影响,我们对各物种miR-153序列进行多序列比对,利用miRanda和TargetScan分析miR-153的靶基因并对其进行功能富集分析,实时荧光定量PCR检测了miR-153和AKT在DBP暴露后大型溞体内的表达变化。结果显示,多序列比对发现11个物种miR-153基因序列保守性很高,20个碱基完全一致;miR-153的742个靶基因,主要富集在DNA模板和转录调控、氧化还原和信号转导等生物学过程,且靶基因参与胞吞、平滑肌收缩、细胞色素P450对异生素的代谢等代谢信号通路的调节;不同浓度、不同时间的DBP暴露后,与对照组相比,大型溞miR-153表达量出现了极显著上升(p < 0.01),AKT基因的表达量与之相反,表现出miR-153和AKT的负调控关系。结果表明,在DBP暴露下miR-153可能通过靶向AKT基因,影响AKT下游相关基因的表达,导致AKT参与的大型溞生命活动失调,本实验为解析DBP对水生动物的毒理机制提供科学参考。

MicroRNAs are involved in the regulation of animal life processes, in-cluding growth, development, biotic and abiotic stress responses. MicroRNAs and target genes have been shown to be involved in the response of aquatic animals to environmental stress. To explore the biological function of miR-153 and the effect of dibutyl phthalate (DBP) exposure on the expres-sion of miR-153 and its target gene AKT in Daphnia magna, we performed alignment of miR-153 sequences of various species. Miranda and Targets can were used to analyze the target gene of miR-153 and perform functional enrichment analysis. The changes of miR-153 and AKT in D. magna after DBP exposure were detected by real-time quantitative PCR. The results showed that the se-quence of miR-153 in 11 species was highly conservative, and 20 bases were completely consistent. The 742 miR-153 targeted genes are mainly concentrated in DNA template, transcriptional regula-tion, redox, signal transduction biological processes. miR-153 targeted genes are involved in the regulation of endocytosis, smooth muscle contraction, cytochrome P450 metabolism of allotropin and other metabolic signaling pathways. After DBP exposure at different concentrations and at dif-ferent times, compared with the control group, the expression of miR-153 in D. magna increased significantly (P < 0.01), and the expression of AKT gene was opposite. It showed a negative regula-tory relationship between miR-153 and AKT. The results showed that miR-153 may affect the ex-pression of AKT downstream-related genes by targeting AKT genes after DBP exposure, leading to an imbalance in the vital activities of D. magna involved in AKT. This experiment provides a scien-tific reference for analyzing the toxicological mechanism of DBP on aquatic animals.