微小RNA及其介导的竞争性内源RNA调控网络在意大利蜜蜂工蜂中肠发育过程中的潜在作用

doi:10.3864/j.issn.0578-1752.2020.12.017

摘要/Abstract

摘要：

【目的】微小RNA（microRNA,miRNA）能够通过靶向结合导致mRNA的抑制或降解,从而对基因表达进行负调控。MiRNA在昆虫的生长、发育、免疫和细胞生命活动调控方面扮演关键角色。本研究旨在对意大利蜜蜂（Apis mellifera ligustica,简称意蜂）工蜂中肠的差异表达miRNA（differentially expressed miRNA,DEmiRNA)及其调控网络进行深入分析,并结合前期在mRNA、长链非编码RNA(long non-coding RNA,lncRNA)和环状RNA(circular RNA,circRNA)组学层面的相关研究结果,系统解析中肠发育过程的miRNA差异表达谱及竞争性内源RNA（competing endogenous RNA,ceRNA）调控网络介导的中肠发育机理。【方法】利用small RNA-seq技术对正常的意蜂7日龄和10日龄工蜂中肠（Am7和Am10）进行测序。将质控后的测序数据比对西方蜜蜂基因组,再将比对上的序列标签（tags）比对到miRBase数据库以鉴定已知miRNA。利用每百万标签序列（tags per million,TPM）算法对miRNA表达量进行计算和归一化处理。根据|log₂fold change|≥1且P≤0.05的标准筛选得到显著性DEmiRNA;利用相关软件进行靶mRNA的预测及GO和KEGG数据库注释。根据靶向结合关系及前期研究结果,利用Cytoscape软件对AMPK、PI3K-Akt、Wnt、cAMP、Hippo、mTOR、Toll/Imd、TGF-beta和MAPK 9条信号通路相关的DElncRNA/DEcircRNA-DEmiRNA-DEmRNA网络进行可视化;构建以miR-342-y为核心的ceRNA调控网络,进一步对网络中的靶mRNA进行代谢通路注释。利用茎环实时荧光定量PCR（Stem-loop RT-qPCR）验证测序数据及miRNA差异表达的可靠性。【结果】Am7 vs Am10比较组中共有112个显著性DEmiRNA,包括38个显著上调miRNA和74个显著下调miRNA,分别靶向结合7 434和9 559个mRNA,它们可注释到生物学进程相关的21和23个功能条目,细胞组分相关的16和17个功能条目,以及分子功能相关的10和11个功能条目;此外还能注释到果糖和甘露糖代谢、嘌呤代谢、甘氨酸、丝氨酸和苏氨酸代谢等物质代谢相关的83和86条通路;内吞作用、泛素蛋白水解、黑色素生成等细胞免疫相关的10和10条通路;MAPK、Jak-STAT、NF-κB等体液免疫相关的5和5条通路;以及Hippo、FoxO、Notch等涉及生长发育的13和11条信号通路。进一步构建上述9条信号通路相关的ceRNA调控网络,分析发现意蜂中肠发育过程中DEmiRNA与DElncRNA、DEcircRNA和DEmRNA之间存在复杂的调控关系;DEmiRNA位于网络中心,而DElncRNA、DEcircRNA和DEmRNA位于网络的外周。miR-342-y在意蜂工蜂中肠和幼虫肠道发育过程中均为显著性下调表达,可靶向结合3个DEcircRNA、4个DElncRNA和327个mRNA。Stem-loop RT-qPCR结果显示4个DEmiRNA的差异表达趋势与测序结果一致,说明本研究中测序数据及miRNA差异表达趋势真实可靠。【结论】DEmiRNA可能通过参与调控物质和能量代谢通路、Hippo和Wnt等信号通路以及细胞和体液免疫通路相关基因的表达影响意蜂中肠的生长和发育;miR-182-x、miR-291-y、miR-342-y、ame-miR-6001-3p等关键DEmiRNA介导的ceRNA调控网络可能在意蜂中肠发育过程发挥重要的调控作用。

关键词: 意大利蜜蜂, 中肠, 微小RNA, 长链非编码RNA, 环状RNA, 竞争性内源RNA, 发育机理

Abstract:

【Objective】 MicroRNA (miRNA) can lead to the inhibition or degradation of mRNA via target binding, thus performing negative regulation of gene expression. MiRNAs play a key role in regulating the growth, development, immunity and cellular activity of insects. This study aims to deeply investigate differentially expressed miRNAs (DEmiRNAs) and their regulatory networks, and to systematically parse differential expression pattern and competing endogenous RNA (ceRNA) mediated mechanism underlying the development of Apis mellifera ligustica worker’s midgut. 【Method】 Midguts of A. m. ligustica 7- and 10-day-old workers were sequenced using small RNA-seq technology. After quality control, the sequencing data were mapped to the genome of A. mellifera, followed by identification of known miRNAs via mapping the mapped tags to miRBase database. Expression levels of miRNAs were calculated and normalized using tags per million (TPM) algorithm. Significant DEmiRNAs were screened out following the criteria of |log₂fold change|≥1 and P≤0.05. Prediction, GO database annotation and KEGG database annotation of target mRNAs were conducted using related software. Based on the target binding relationship and previous findings, DElncRNA/DEcircRNA-DEmiRNA-DEmRNA networks associated with 9 signaling pathways (AMPK, PI3K-Akt, Wnt, cAMP, Hippo, mTOR, Toll/Imd, TGF-beta and MAPK signaling pathways) were visualized using Cytoscape software. CeRNA regulatory network of miR-342-y was further constructed followed by KEGG pathway annotation of target mRNAs involved in the network. Stem-loop RT-qPCR was used to verify the reliability of our sequencing data and differential expression of miRNAs. 【Result】 A total of 112 significant DEmiRNAs were identified in Am7 vs Am10 comparison group, including 38 up-regulated miRNAs and 74 down-regulated miRNAs, which could respectively target 7 434 and 9 559 mRNAs. These target mRNAs could be annotated to 21 and 23 functional terms associated with biological process, 16 and 17 terms associated with cellular component, and 10 and 11 terms associated with molecular function. Additionally, the targets could be annotated to 83 and 86 pathways related to material metabolisms such as fructose and mannose metabolism, purine metabolism, glycine, serine and threonine metabolism; 10 and 10 pathways related to cellular immune such as endocytosis, ubiquitin mediated proteolysis and melanogenesis; 5 and 5 pathways related to humoral immune such as MAPK, Jak-STAT and NF-κB; 13 and 11 signaling pathways related to development such as Hippo, FoxO and Notch. Furthermore, ceRNA regulatory networks of aforementioned 9 signaling pathways were constructed and analyzed, the result showed the existence of complex relationship among DEmiRNAs, DElncRNAs, DEcircRNAs and DEmRNAs; DEmiRNAs were located in the center, while DElncRNAs, DEcircRNAs and DEmRNAs were around the network. Further investigation suggested that miR-342-y was significantly down-regulated in the developmental processes of both worker’s midgut and larval gut of A. m. ligustica; this miRNA could target 3 DEcircRNAs, 4 DElncRNAs and 327 mRNAs. Stem-loop RT-qPCR result indicated that the differential expression trend of 4 DEmiRNAs was in accordance of that in sequencing result, confirming the authenticity of the sequencing data and differential expression pattern of miRNAs. 【Conclusion】 DEmiRNAs are likely to affect the growth and development of A. m. ligustica worker’s midgut by participating in regulation of the expression of genes engaged in material and energy metabolism-associated pathways, signaling pathways such as Hippo and Wnt as well as cellular and humoral immune pathways; ceRNA regulatory networks mediated by several key DEmiRNAs including miR-182-x, miR-291-y, miR-342-y and ame-miR-6001-3p are likely to play pivotal regulatory parts during the developmental process of the midgut of A. m. ligustica.

Key words: Apis mellifera ligustica, midgut, microRNA (miRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), competing endogenous RNA (ceRNA), developmental mechanism

杜宇,范小雪,蒋海宾,王杰,范元婵,祝智威,周丁丁,万洁琦,卢家轩,熊翠玲,郑燕珍,陈大福,郭睿. 微小RNA及其介导的竞争性内源RNA调控网络在意大利蜜蜂工蜂中肠发育过程中的潜在作用[J]. 中国农业科学, 2020, 53(12): 2512-2526.

DU Yu,FAN XiaoXue,JIANG HaiBin,WANG Jie,FAN YuanChan,ZHU ZhiWei,ZHOU DingDing,WAN JieQi,LU JiaXuan,XIONG CuiLing,ZHENG YanZhen,CHEN DaFu,GUO Rui. The Potential Role of MicroRNAs and MicroRNA-Mediated Competing Endogenous Networks During the Developmental Process of Apis mellifera ligustica Worker’s Midgut[J]. Scientia Agricultura Sinica, 2020, 53(12): 2512-2526.

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引物名称Primer name	引物序列Primer sequence (5′-3′)
Loop-miR-210-z	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACAGCCGC
F-miR-210-z	ACACTCCAGCTGGGCTGTGCGTGTGACA
Loop-miR-342-y	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACGGGTGC
F-miR-342-y	ACACTCCAGCTGGGTCTCACACAGAAATC
Loop-miR-7975-y	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTGGTGCCG
F-miR-7975-y	ACACTCCAGCTGGGATCCTGGTCA
Loop-miR-155-x	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACCCCTA
F-miR-155-x	ACACTCCAGCTGGGTTAATGCTAATTGTGA
R	CTCAACTGGTGTCGTGGA
U6-F	GTTAGGCTTTGACGATTTCG
U6-R	GGCATTTCTCCACCAGGTA

样品 Sample	原始读段 Raw reads	有效读段 Clean reads
Am7-1	14021728	13619226 (97.13%)
Am7-2	13479635	13081146 (97.04%)
Am7-3	13033478	12656635 (97.11%)
Am10-1	13083648	12577875 (96.13%)
Am10-2	13544596	13137501 (96.99%)
Am10-3	13716280	13316876 (97.09%)

差异表达miRNA ID DEmiRNA ID	以2为底miRNA的相对变化倍数的对数值 log₂fold change	P值 P value
miR-7132-y	13.27	8.68E-09
miR-2188-x	12.72	2.31E-06
miR-1388-x	12.43	6.78E-06
miR-1338-x	12.38	7.39E-06
miR-1388-y	11.76	4.20E-04
miR-1338-y	11.67	9.18E-04
miR-7132-x	11.16	9.01E-03
miR-3964-y	11.09	3.37E-03
miR-7935-y	11.01	4.31E-03
miR-6537-x	10.46	0.023

差异表达miRNA ID DEmiRNA ID	以2为底miRNA的相对变化倍数的对数值 log₂fold change	P值 P value
miR-8503-x	-14.09	3.09E-04
miR-298-x	-13.64	2.65E-08
miR-291-y	-12.94	8.67E-05
miR-292-y	-12.75	3.40E-04
miR-4700-x	-12.55	0.015
miR-293-y	-12.27	6.77E-04
miR-2965-y	-12.13	0.030
miR-2518-y	-12.13	6.13E-03
miR-300-y	-12.03	2.96E-04
miR-4577-y	-12.01	0.038

通路 Pathway	通路ID Pathway ID	靶mRNA富集数 Number of target mRNAs	P值 P value
钙信号通道 Calcium signaling pathway	ko04020	77	1.30E-14
催产素信号通路 Oxytocin signaling pathway	ko04921	82	1.07E-14
坏死性凋亡 Necroptosis	ko04217	58	5.72E-16
嗅觉传导 Olfactory transduction	ko04740	50	3.01E-17
长时程增强 Long-term potentiation	ko04720	70	3.98E-18
Hippo信号通路 Hippo signaling pathway	ko04390	118	1.29E-18
促性腺激素释放激素信号通路 GnRH signaling pathway	ko04912	79	4.76E-19
神经营养因子信号通路 Neurotrophin signaling pathway	ko04722	89	3.17E-21
胆碱能突触 Cholinergic synapse	ko04725	79	2.90E-21
胃酸分泌 Gastric acid secretion	ko04971	69	2.05E-21
醛固酮合成与分泌 Aldosterone synthesis and secretion	ko04925	74	1.94E-21
cAMP信号通路 cAMP signaling pathway	ko04024	113	1.73E-21
昼夜节律调节 Circadian entrainment	ko04713	75	2.93E-23
胰岛素分泌 Insulin secretion	ko04911	78	2.53E-23
ErbB信号通路 ErbB signaling pathway	ko04012	80	2.43E-25