The role of miRNAs and lncRNAs in regulating transcription in gilthead sea bream (Sparus aurata) myoblasts in response to amino acids and insulin-like growth factor 1 [RNA-seq]

In this study, gilthead sea bream (Sparus aurata) fast muscle myoblasts were stimulated with two pro-growth treatments, amino acids (AA) and insulin-like growth factor 1 (Igf-1), to analyze the transcriptional response of genes, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and their regulatory network. AA had a higher impact on gene transcription (1795 genes significantly changed) compared to Igf-1 (385 genes significantly changed). Both treatments stim-ulated the transcription of genes related to muscle differentiation (GO:0042692) and sarcomere components (GO:0030017), but AA stimulated more the DNA replication and cell division (GO:0007049). Notably, four miRNAs (miR-21, miR-146, miR-22b and miR-206) dominated the landscape among 403 expressed miRNAs. Both pro-growth treatments altered the transcription of over 100 miRNAs, including muscle-specific miRNAs (myomiRs) such as miR-133a/b, miR-206, miR-499, miR-1, and miR 27a. Among 111 detected lncRNAs (> 1 FPKM), only 30 were significantly changed by AA and 11 by Igf-1. Eight lncRNAs exhibited strong negative correlations with several mRNAs, suggesting direct regulation; while 30 lncRNAs showed strong correlations and interac-tions with several miRNAs, suggesting their function as miRNA’s sponges. This work is the first step in the identification of ncRNAs network controlling muscle development and growth in gilthead sea bream, pointing out potential regulatory mechanisms in response to pro-growth signals. To obtain the transcriptional profile of gilthead sea bream (Sparus aurata) muscle cells treated with amino acids (AA), IGF1 or cultured in nutrient restriction medium (Control), with n=3 for each experimental group.

本研究以金头鲷（Sparus aurata）快肌成肌细胞为实验材料，采用两种促生长处理方式——氨基酸（Amino acids, AA）与胰岛素样生长因子1（Insulin-like growth factor 1, Igf-1）进行刺激，以分析基因、微小RNA（microRNAs, miRNAs）及长链非编码RNA（long non-coding RNAs, lncRNAs）的转录响应及其调控网络。相较于胰岛素样生长因子1（Igf-1）处理组（仅385个基因发生显著表达变化），氨基酸（AA）处理对基因转录的影响更为显著，共1795个基因出现显著表达改变。两种促生长处理均能激活与肌肉分化（GO:0042692）及肌节组分（GO:0030017）相关的基因转录，但氨基酸（AA）处理更显著促进了DNA复制与细胞分裂（GO:0007049）相关通路的基因表达。值得注意的是，在403个可检测到的微小RNA（miRNAs）中，miR-21、miR-146、miR-22b及miR-206这4种miRNAs占据了表达谱的主导地位。两种促生长处理均能改变超过100种miRNAs的转录水平，其中包括肌肉特异性miRNAs（肌源性miRNAs, myomiRs），如miR-133a/b、miR-206、miR-499、miR-1及miR-27a。在111个检测到的长链非编码RNA（lncRNAs）中（FPKM>1），仅30个可被氨基酸（AA）处理诱导显著表达变化，11个可被胰岛素样生长因子1（Igf-1）处理诱导显著改变。8个lncRNAs与多种mRNA呈现显著负相关，提示其可能直接参与靶基因的转录调控；另有30个lncRNAs可与多种miRNAs形成强相关互作网络，表明其可能作为miRNA海绵发挥调控功能。本研究首次鉴定了金头鲷（Sparus aurata）中调控肌肉发育与生长的非编码RNA（non-coding RNAs, ncRNAs）调控网络，阐明了其响应促生长信号的潜在调控机制。本实验设置3个实验组：氨基酸（AA）处理组、胰岛素样生长因子1（IGF1）处理组及营养限制培养对照组（Control），每组设置3个生物学重复，以获取各组金头鲷肌肉细胞的转录组谱。