Identification And Functional Verification Of Long Non-Coding RNAs In The Fetal Longissimus Dorsi Muscle Development Of Goat

Meat production traits as an important economic trait affecting the production efficiency of meat-type goats,it has always been a hot topic in the field of meat goats genetics and breeding.Molecular regulation of muscle growth is a breakthrough in the improvement of molecular breeding of meat goats.Skeletal muscle growth and development of goat are regulated by various regulatory factors.Previous studies have focused on DNA,mRNA,and miRNA levels,and the regulation of lncRNA on growth and development of goat skeletal muscle,especially goat fetus has not been reported.Here in this study,longissimus dorsi muscle samples were collected at these four developmental stages: three gestation stages?45,60,105 days of gestation,denoted as E45,E60,and E105,respectively?and one neonatal stage?the three –day-old newborn,denoted as B3?from Jianzhou big-eared goats.By using RNA sequencing?RNA-seq?approach,a large number of lncRNA transcripts were identified and its genomic characteristics are analyzed.The differential expressed lncRNAs between the different developmental stages of goat skeletal muscle were identified and functional prediction was performed.The mechanism of lnc₆810 promoting the differentiation of goat skeletal muscle satellite cells?SMSCs?was analyzed at the cellular level.The main results as follows.1.We performed large-scale cDNA sequencing experiments in eleven libraries using the Illumina paired-end RNA-seq approach,resulting in a total of ¹31 Gb?gigabases?paired-end reads in 125 nucleotide?nt?length.An average of 11.97 ± 1.87 Gb of raw reads was obtained per library.And about 98.20% of the reads passed initial quality thresholds?Illumila Protocol?and were deemed as clean reads for the subsequent analyses.Approximately 78.99-87.73% of the clean reads in all the libraries were mapped to the goat reference genome?NCBI,CHIR₁.0?by using TopHat2 software,and about 78.52%-86.60% of the clean reads have a unique genomic location.We obtained an average of 51,053?48,398⁵3,266?transcripts for each sequencing library.On average,which covered 80.41%?75.88%⁸5.10%?of goat reference transcripts,and the transcripts with the coverage of 90% or more in each library account for 51.44% of the total transcripts.2.A total of 3,981 reliable lncRNA transcripts were identified,including 3,515 intergenic lncRNAs and 466 antisense lncRNAs.Compared to protein coding genes,the lncRNAs identified in this study have much lower exon number,shorter transcript length,and lower protein coding potential.A total of 577 differentially expressed lncRNAs were identified across four stages based on pairwise comparisons.We did not detect any stage-specific expressed lncRNAs among the four developmental stages,but 154 differentially expressed lncRNAs were observed in all four developmental stages.3.In functional prediction of lncRNA,through GO and KEGG pathway analysis of cis and trans target genes of lncRNA,we found that many protein-coding genes are enriched in transcriptional regulation and muscle development-related biological process GO terms and signaling pathways,indicating that lncRNA may be involved in the biological process of muscle development by influencing the expression of these protein-encoding genes.4.The lncRNA expression results from RNA-seq were verified by qPCR,we found the two quantitative results showed the same expression trend in different developmental stages,and have a good correlation?r = 0.94⁰.98?with each other,indicating that the RNA-seq results are accurate and reliable.5.Through comprehensive analysis of RegRNA 2.0 software and miRBase database,it is predicted that there may be a binding site of chi-miR-125 b on the sequence of lnc₆810.We used a dual luciferase reporter assay to analyze the interaction of lnc₆810 and chi-miR-125 b.Compared with the control cells,the cells transfected with miR-125 b mimics and wild type recombinant plasmid had a significantly lower luciferase activity,but the cells transfected with miR-125 b mimics and mutant recombinant plasmid had no significant effect on luciferase activity.These results indicated that lnc₆810 is the target gene of chi-miR-125 b.Fluorescence in situ hybridization assay showed that lnc₆810 was mainly located in the cytoplasm.6.In the process of goat SMSCs differentiation,the expression of lnc₆810 showed an up-regulated trend,while the expression of miR-125 b showed a down-regulated trend.After lnc₆810 overexpression,the expression of miR-125 b was down-regulated,while the expression of MyoD,MyoG and IGF-2 genes were up-regulated.After the expression of lnc₆810 was interfered,the expression of miR-125 b was up-regulated,while the expression of MyoD,MyoG and IGF-2 genes were down-regulated.Theses results indicated that lnc₆810 can bind miR-125 b by acts as a competing endogenous RNA,alleviate the inhibitory effect of miR-125 b on its target gene IGF-2,thereby increasing the expression of IGF-2 gene in cells and promoting the differentiation of goat SMSCs.7.Through overexpression and inhibition experiment of miR-125 b,we found that after miR-125 b overexpression,the expression of MyoD,MyoG and IGF-2 genes were down-regulated,while the expression of lnc₆810 was down-regulated.After the expression of miR-125 b was inhibited,the expression of MyoD,MyoG and IGF-2 genes were up-regulated,while the expression of lnc₆810 was up-regulated.Theses results indicated that miR-125 b can inhibit the differentiation of SMSCs,and also proved that lnc₆810 can play a functional role in the differentiation of goat SMSCs by acting as a competing endogenous RNA.This study provides a reference for further revealing the molecular mechanisms of goat skeletal muscle development by identifying the lncRNAs involved in development of skeletal muscle and analyzing lnc₆810-miR-125b-IGF2 regulatory networks.