东方蜜蜂微孢子虫孢子中长链非编码RNA的竞争性内源RNA调控网络及潜在功能

Investigation of Competing Endogenous RNA Regulatory Network and Putative Function of Long Non-Coding RNAs in Nosema ceranae Spore

[背景]长链非编码RNA(long non-coding RNA,lncRNA)是一类长度>200 nt且不具有编码能力的转录本,能够在真核生物的转录水平和转录后水平通过顺式(cis)、反式(trans)或竞争性内源RNA(competing endogenous RNA,ceRNA)机制调控基因的表达,已被证明在生物体的生长和发育等生物学过程中发挥重要的调控作用.[目的]结合测序得到的东方蜜蜂微孢子虫(Nosema ceranae)纯化孢子的small RNA(sRNA)组学数据和前期获得的lncRNA组学数据对lncRNA的调控方式进行深入细致的分析和探讨,揭示lncRNA在东方蜜蜂微孢子虫孢子中的潜在功能.[方法]利用small RNA-seq(sRNA-seq)技术对东方蜜蜂微孢子虫纯化孢子进行测序,通过相关的生物信息学软件对测序数据进行质控.根据lncRNA与mRNA的位置关系预测lncRNA的上下游基因;使用Blast软件比对GO和KEGG数据库,对上下游基因进行功能和代谢通路注释;利用Target Finder软件预测lncRNA-miRNA及lncRNA-miRNA-mRNA调控网络,进而通过Cytoscape v3.7.2软件对调控网络进行可视化;利用RT-PCR对调控网络中的部分lncRNA、miRNA和mRNA的表达进行验证.[结果]东方蜜蜂微孢子虫纯化孢子样品的sRNA-seq分别得到16597883、15451791和12248316条raw reads,经质控分别得到15608370、14249255和11440684条clean reads,各样品的Q30≥98.58%.共预测出lncRNA的上下游基因310个,它们可注释到代谢进程、细胞进程、催化活性、结合和细胞等相关的35个功能条目;此外还可注释到56条代谢通路,涉及嘌呤代谢、碳代谢和丙酮酸代谢等物质代谢通路,以及甲烷代谢、氧化磷酸化和糖酵解/糖异生等能量代谢通路.上述结果表明相应的lncRNA可能通过cis作用调节上下游基因的表达,从而参与调控东方蜜蜂微孢子虫孢子中的物质和能量代谢以及细胞生命活动.进一步构建lncRNA-miRNA调控网络,分析结果显示MSTRG.3636.1、MSTRG.4498.1和MSTRG.4883.1可靶向结合4个miRNA(nce-miR-7729、nce-miR-7502、nce-miR-8639和nce-miR-8565),说明此3个lncRNA可作为ceRNA在东方蜜蜂微孢子虫孢子中发挥潜在作用.LncRNA-miRNA-mRNA调控网络分析结果显示,三者之间形成较为复杂的调控网络关系,miRNA处于调控网络的中心并联系lncRNA和mRNA,nce-miR-7502和nce-miR-8639结合的mRNA最多,达到28个;与MSTRG.3636.1、MSTRG.4498.1和MSTRG.4883.1存在靶向关系的miRNA结合多个mRNA,表明上述3个lncRNA可能通过ceRNA机制发挥作用,从而影响东方蜜蜂微孢子虫孢子中的新陈代谢和生命活动.[结论]lncRNA可能通过cis作用调控上下游基因的表达,以及作为ceRNA通过吸附miRNA间接影响靶基因的表达,从而参与调节东方蜜蜂微孢子虫孢子中的物质和能量代谢等基本生命活动.

【Background】Long non-coding RNAs(lncRNAs)are a kind of non-coding transcripts with a length of more than 200 nt.LncRNAs can regulate gene expression at transcriptional level and post-transcriptional level via cis effect,trans effect or competing endogenous RNA(ceRNA)mechanism.LncRNAs have been proved to play pivotal roles in many biological processes such as growth and development.【Objective】The objective of this study is to perform detailed investigation and discussion of the regulatory manner of lncRNAs by combining small RNA(sRNA)omics dataset obtained in this work and previously gained lncRNA omics dataset,and to reveal the putative function of lncRNAs in Nosema ceranae spores.【Method】Here,purified spores of N.ceranae were sequenced using small RNA-seq(sRNA-seq),related bioinformatics software was used to conduct quality control of sequencing data.The upstream and downstream genes of N.ceranae lncRNAs were predicted based on the positional relationship between lncRNAs and mRNAs.Annotations of these upstream and downstream genes in GO and KEGG databases were carried out using Blast software.The lncRNA-miRNA and lncRNA-miRNA-mRNA regulatory networks were predicted using Target Finder software and then visualized with Cytoscape v3.7.2 software.The expression of partial lncRNAs,miRNAs and mRNAs within regulatory networks was validated with RT-PCR.【Result】In total,16597883,15451791 and 12248316 raw reads were obtained from sRNA-seq of N.ceranae spore samples,respectively,and after quality control,15608370,14249255 and 11440684 clean reads with a mean Q30 above 98.58%were gained,respectively.A total of 310 upstream and downstream genes of lncRNAs were predicted.These genes could be annotated to 35 functional terms associated with metabolic process,cell process,catalytic activity,binding and cells,etc.Additionally,these genes could be annotated to 56 metabolic pathways,including material metabolic pathways such as purine metabolism,carbon metabolism,pyruvate metabolism;and energy metabolic pathways including methane metabolism,oxidative phosphorylation,glycolysis/gluconeogenesis and so forth.The results indicated that corresponding lncRNAs could regulate the expression level of upstream and downstream genes through cis effect,thus participating in regulation of material and energy metabolisms as well as cell activities in N.ceranae spore.Furthermore,lncRNA-miRNA regulatory networks were constructed and analyzed,the result showed that MSTRG.3636.1,MSTRG.4498.1 and MSTRG.4883.1 could bind to four miRNAs including nce-miR-7729,nce-miR-7502,nce-miR-8639 and nce-miR-8565,suggesting that these three lncRNAs as ceRNAs could exert potential function in N.ceranae spore.LncRNA-miRNA-mRNA regulatory network analysis demonstrated that complex networks existed among them,miRNAs were located in the center,connecting lncRNAs and mRNAs,nce-miR-7502 and nce-miR-8639 could bind to the most mRNAs(28).MiRNAs targeting MSTRG.3636.1,MSTRG.4498.1 and MSTRG.4883.1 could target several mRNAs,indicating that these three lncRNAs might play a role via ceRNA mechanism,thus affecting the mechanism and vital activity in N.ceranae spore.【Conclusion】LncRNAs are likely to regulate the expression of upstream and downstream genes via cis effect,and indirectly affect the expression of target genes by absorbing miRNAs as ceRNAs,thus controlling basic activities in N.ceranae spore such as material metabolism and energy metabolism.