LncRNAs and their regulatory networks in breast muscle tissue of Chinese Gushi chickens during late postnatal development

- However, studies on the role of lncRNAs in the late stages of chicken breast muscle development are still lacking.
- Furthermore and 78 DE-lncRNAs were found in the W14 vs.
- After GO enrichment analysis of the DE-lncRNAs, several muscle development-related GO terms were found in the W22 vs.
- Moreover, it was found that the MAPK signaling pathway was one of the most frequently enriched pathways in the different comparison groups.
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- Several candidate genes, such as the growth hormone se- cretagogue receptor (GHSR) [5], insulin-like growth fac- tors (IGFs) [6], transforming growth factor beta 2 (TGFβ2) [7], and myocyte enhancer factor 2B (MEF2B) [8], have been identified to play important roles in the growth of chickens.
- Although many genes play important roles in chicken muscle growth, studies have shown that only a small percentage (1–2%) of the genome encodes proteins in mammals, and tens of thousands of intergenic sites are transcribed into noncoding RNA [9].
- In the past few years, regulatory RNAs, such as miRNAs, piRNAs, snoRNAs, and long noncoding RNAs (lncRNAs), have appeared to play roles in many important biological processes [10].
- In complex organisms, lncRNAs contain hidden regulatory information that can play a role in the regulation of gene expression [11].
- For example, as one of the earliest identi- fied lncRNAs, H19 also plays a regulatory role in various growth and development processes [13].
- For ex- ample, lncIRS1 acts as a sponge of the miR-15 family, regulating the expression of insulin receptor substrate 1 (IRS1), thereby promoting skeletal muscle production [18].
- Our previous histological study of this type of breast muscle showed that before 22 weeks of age, muscle fiber diameter grew rapidly, and after 22 weeks of age, the relationship between the diameter and density of the breast muscle fibers remained balanced [19].
- To generate a complete annotation of the noncoding transcriptome of the Gushi chicken breast muscle tissue beyond the currently annotated transcriptome, we first used Cuff- merge [20] to combine and then screen the transcripts.
- d, e, f Distribution of transcript lengths, distribution of the number of exons per transcript, and distribution of the number of ORFs (mRNA: green, annotated lncRNA: purple, and novel lncRNAs: red).
- Among the six different comparison groups, there were and 78 DE-lncRNAs in the W14 vs.
- Only LNC_000920 was commonly found in the follow- ing five comparison groups: W14 vs.
- Moreover, LNC_000255 appeared in the following comparison groups: W14 vs.
- It is possible that from 14 weeks - 22 weeks, many of the same lncRNAs played a common role, which also led to the reduc- tion in DE-lncRNA that was commonly seen in the six comparison groups.
- Moreover, after identifying DE-lncRNAs, we analyzed the chromosome distribu- tion information of the DE-lncRNAs and found that DE-lncRNAs were distributed in almost all chromo- somes but not in chromosomes 22 and 27, and the largest number was found in chromosome 1 (Fig.
- The lncRNA expression level was determined and showed a similar pattern to that of the RNA-seq data (Fig.
- To investigate the possible functions of the DE- lncRNAs in breast muscle between the different de- velopmental stages, we conducted Gene Ontology (GO, http://www.geneontology.org/) enrichment ana- lysis to uncover the enriched biological process terms associated with DE-lncRNA-targeted DEGs for each comparison group.
- enrichment analysis of the cis-targets of lncRNAs showed that only one growth- and development- related GO term, called positive regulation of embry- onic development, was found in the W22 vs.
- In the GO analysis of the trans-targets of lncRNAs, we found several muscle development-related GO terms only in the W22 vs.
- In the KEGG pathway analysis of the cis-targets of lncRNAs, the phago- some pathway was identified as the most significantly enriched pathway for the W14 vs.
- Moreover, the KEGG pathway analysis of the trans-targets of lncRNAs showed that the propanoate metabolism pathway and fatty acid metabolism pathway were the top two pathways for.
- In the W22 vs.
- We found that the MAPK signaling pathway was one of the most frequently enriched pathways in both the W22 vs.
- To explore how lncRNAs interact with their target genes to regulate chicken muscle development and to identify key molecular players in the process, we first predicted the cis- and trans-targets of DE-lncRNAs and then con- structed the regulatory networks between DE-lncRNAs and their target genes.
- 4 The enriched GO terms of the DE-lncRNA.
- a Cis-target genes in the W22 vs.
- b Trans-target genes in the W22 vs..
- 5 The enriched KEGG pathways of the DE-lncRNA.
- a Trans-target genes in the W22 vs.
- b Trans-target genes in the W30 vs.
- In the networks of cis-target DEGs of DE-lncRNAs of the muscle development-related GO terms, we found a total of 10 interaction relationships between 3 genes and 10 lncRNAs (Fig.
- In addition, in the networks of trans- target DEGs of DE-lncRNAs of the muscle development-related GO terms, we found 7 interaction networks between 3 genes and 7 lncRNAs (Fig.
- Fur- thermore, we also generated the lncRNA-mRNA net- works of the frequently enriched MAPK signaling pathway, which had a total of 25 interaction relation- ships between 11 genes and 25 cis-regulating lncRNAs and 27 interaction relationships between 8 genes and 17 trans-regulating lncRNAs (Fig.
- Interestingly, we found that the networks containing MEF2C and its tar- geting lncRNAs (ALDBGALT ALDB- GALT LNC_001247) were not only in the muscle development-related GO terms but also in the MAPK signaling pathway..
- To identify potential ceRNA networks in the development of chicken breast muscle, we constructed ceRNA networks of DEGs, differentially expressed miRNAs (DEMs), and DE- lncRNAs (q-value <.
- For example, 445 ceRNA networks were found in the lncRNA-miRNA- mRNA network in the W14 vs.
- Moreover, there were 450 ceRNA networks in the W30 vs.
- The PPI network from DE-lncRNA cis-target genes of the W14 vs.
- Moreover, in the W22 vs.
- However, no PPI net- work was found in the DE-lncRNA trans-target genes of.
- 0.98) in the MAPK signaling pathway (The network relations were refer to the KEGG website: https://www.genome.jp/kegg/pathway.html.
- In genetics research, previous studies focused on the role of protein-coding genes in skeletal muscle growth and development, and it was later found that noncoding RNA represents the majority of the tran- scriptome, with studies showing that only <.
- In the present study, functional enrichment analyses of both cis- and trans-target genes of DE-lncRNAs revealed that the muscle development-related enriched GO terms were found only in the W22 vs.
- This is consistent with our previous transcriptomics ana- lysis [19] and indicates that the period from 14 to 22 weeks is an important stage in the development of chicken breast muscle.
- In addition, in the pathway en- richment analysis of the DE-lncRNA trans-target genes,.
- 9 The lncRNA-miRNA-mRNA ceRNA networks of muscle development-related GO terms.
- (a) Cis-target genes of the W14 vs.
- (b) Cis-target genes of the W22 vs..
- (c) Cis-target genes of the W30 vs.
- (D) Trans-target genes of the W30 vs.
- it was found that there was a common pathway in the W22 vs.
- We speculate that the DE-lncRNAs in the W22 vs..
- Thus, we constructed interaction networks of DE-lncRNAs and their cis- and trans-target genes, and only some of the genes associated with muscle development-related GO terms and the MAPK signaling pathway and their corresponding lncRNAs are shown in Fig.
- Interestingly, we found that the networks contain- ing MEF2C and its targeting lncRNAs were not only in the muscle development-related GO terms but also in the MAPK signaling pathway.
- miR-140 binding sites were identified in NEAT1, and the authors found that mature miR-140 could physically interact with NEAT1 in the nucleus, thereby promoting the ex- pression of NEAT1 [34].
- Therefore, we speculate that the target lncRNAs of these miRNAs may also be in- volved in the muscle development process.
- For example, in the W14 vs.
- Furthermore, the ceRNA net- work analysis showed that ANKRD1 is involved in 13 ceRNA networks containing two miRNAs (miR-148a-3p and miR-10b-5p) and 12 lncRNAs in the W14 vs.
- Moreover, ANKRD1 was in- volved in 8 ceRNA networks containing gga-miR-92-3p with 8 lncRNAs in the W30 vs.
- The interaction between IGF-I and epider- mal growth factor (EGF) was identified in the W14 vs..
- It has been reported that the ex- pression of the IGF gene is enhanced during muscle hypertrophy and that locally produced IGF-I may play roles in skeletal muscle growth [43].
- The WNT/planar cell polarity (PCP) sig- naling pathway is involved in the development of human cancer.
- In addition, some of the DE-lncRNA trans-targets from the W30 vs.
- W22 comparison group were included in the CDK8-CCNC PPI network.
- Therefore, the interactions between these genes may eventually affect muscle development, and the above results indicate that there are complex intergenic interactions in the development of chicken breast muscle..
- W14 comparison groups, some GO terms related to muscle development were found, further indicating that between 14 and 22 weeks, changes in the expression of some crucial lncRNAs and their target genes affected the growth and development of chicken breast muscles during these important stages..
- protein and 12.35 MJ/kg were prepared in the first stage (younger than 14 weeks) and 15.6% crude protein and 12.75 MJ/kg were prepared in the second stage (older than 14 weeks), and the chickens had free access to water.
- Chickens were anesthetized by intravenous injec- tion of sodium pentobarbital (40 mg/kg) at a concentra- tion of 0.2% in the wing vein.
- The raw data in the fastq format were first processed by in-house scripts.
- An index of the reference genome was built using Bowtie v2.2.3, and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12.
- the third step was to use Cuffcompare software to filter out the transcripts that overlapped with exon re- gions annotated in the database, and the lncRNAs in the database that overlapped with the exon regions of spli- cing transcripts were annotated as lncRNAs in subse- quent analyses.
- In this study, the resulting transcripts with no protein-coding potential in the software analyses resulted in the lncRNA dataset, and the transcripts that were predicted to have protein- coding potential by at least one coding potential predic- tion software were set as TUCPs..
- Based on Illumina sequencing data, FPKM values were used to assess the expression levels of lncRNAs in the li- braries constructed from breast muscle.
- In the present study, DEGs located ∼100 kb upstream and downstream of DE- lncRNAs were classified as cis-acting target genes.
- All potential target genes for DE-lncRNAs in each comparison group were used in the bioinformatics ana- lysis.
- The network of interac- tions between these DEGs was generated in the STRING database and imported into Cytoscape software for visualization.
- Statistical significance of the qRT-PCR quantitative expression data was tested by per- forming two-tailed unpaired t-tests [48].
- The enriched GO terms of the DE-lncRNA.
- (A- B) Cis-target genes in the W14 vs.
- (C-D) Trans-target genes in the W14 vs.
- The enriched KEGG pathways of the DE- lncRNA.
- (A-C) Cis-target genes in the W14 vs.
- (D) Trans-target genes in the W14 vs.
- DE- lncRNAs: Differentially expressed lncRNAs.
- The funding bodies had no role in the study design or in any aspect of the.
- collection, analysis and interpretation of the data or in the writing of the manuscript..
- All raw sequences have been deposited in the NCBI database Sequence Read Archive with the accession numbers PRJNA516810 (mRNA and lncRNA) and PRJNA516961 (miRNA)..
- Integrated analysis of Long non-coding RNAs (LncRNAs) and mRNA expression profiles reveals the potential role of LncRNAs in skeletal muscle development of the chicken.
- H19 acts as a trans regulator of the imprinted gene network controlling growth in mice.
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- Baker AH: lncRNA/MicroRNA interactions in the vasculature.
- DYNLL/LC8: a light chain subunit of the dynein motor complex and beyond.
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