低氧胁迫下鲢miR-17a-5p及其靶基因分析

Analysis of miR-17a-5p and its target genes in Hypophthalmichthys molitrix under hypoxia stress

为了探究miR-17a-5p在鲢低氧胁迫下的功能,在前期鲢small RNA测序的基础上对鲢miR-17a-5p进行靶基因预测及功能富集分析,通过双荧光素酶活性验证其与HIF-1α的靶向关系,并检测低氧胁迫下miR-17a-5p和其靶基因在鲢肝脏、脑、心脏和鳃四个组织中的动态表达特征。结果显示,鲢miR-17a-5p在不同物种间高度保守,预测出381个miR-17a-5p的潜在靶基因显著富集在硫代谢、mTOR信号通路以及萜类骨架的生物合成3个KEGG信号通路上。miR-17a-5p可与HIF-1αmRNA的3′UTR结合,并降低HIF-1αmRNA水平,低氧胁迫下miR-17a-5p的表达呈下降趋势,而HIF-1α表达则呈上升趋势;3个显著富集KEGG通路中的11个miR-17a-5p潜在靶基因在低氧胁迫过程中的不同组织中的表达,尤其是肝脏中的表达,除SGK1外,均呈显著上升趋势。研究表明,低氧胁迫下鲢各组织中miR-17a-5p的表达下调减弱了其对靶基因的抑制作用,进而导致HIF-1α、ddit4和Lrp5等响应低氧胁迫的基因表达上调。本研究为低氧胁迫下鲢miRNA的表达与调控机制提供新见解,也为培育耐低氧的鲢新品系(种)提供了参考。

Silver carp(Hypophthalmichthys molitrix)is an important freshwater economic fish in China’s aquaculture industry.In 2020,six species of freshwater farmed fish in China had production greater than a million tons,of which the yield of silver carp was 3.8129 million tons,ranking second.Silver carp is widely distributed in natural waters,and plays a major role in regulating the ecological environment.In the process of H.molitrix culture,hypoxia has become an important factor restricting the increase of its yield.In previous studies,miR-17a-5p was identified and differentially expressed in H.molitrix under hypoxia stress based on the small RNA sequencing.The micro-ribonucleic acids(miRNAs),a class of endogenous and noncoding small RNA,play an important role in biological resistance to environmental stresses.In order to explore the role of miR-17a-5p in H.molitrix under hypoxia,sequence analysis and target gene prediction of miR-17a-5p was performed.The results showed that the miR17a-5p are highly conserved among species,and 381 target genes were identified,including HIF-1α.Dual-luciferase reporter assay results demonstrated that when 293T cells were co-transfected with miR17a-5p mimics and WT-pmirGLO-HIF-1α3′UTR,the luciferase activity decreased,confirming HIF-1αto be a target gene of miR17a-5p.This conclusion is in agreement with the observation of the increasing trend of HIF-1αalongside the decreasing pattern of miR17a-5p under hypoxic stress in different tissue.KEGG pathway analysis revealed that the target genes were significantly enriched in Sulfur metabolism,mTOR signaling pathway and Terpenoid backbone biosynthesis.The expression of 11 target genes involved in the above three signaling pathways under hypoxia in liver,brain,heart and gill were detected by quantitative real-time PCR.The results showed that the overall trend of increase for the relative expressions of these genes,and upregulation was most pronounced in liver.In this study,we analyzed the bioinformatics of miR-17a-5p,predicted and further identified its target genes,and subjected H.molitrix to different degree of hypoxia stress,revealing that the downregulation of miR-17a-5p expression in H.molitrix under hypoxia stress attenuates its inhibitory effect on its target genes.Our finding provides a new idea for miR-17a-5p regulation of its target genes in H.molitrix under hypoxia stress,and provides a reference for further analysis of the role of miRNAs in the process of hypoxia tolerance of fish.