Systematic transcriptome-wide analysis of mRNA-miRNA interactions reveals the involvement of miR-142-5p and its target (FOXO3) in skeletal muscle growth in chickens.

The goal of this study was to perform a systematic transcriptome-wide analysis of mRNA-miRNA interactions and to identify candidates involved in the interplay between miRNAs and mRNAs that regulate chicken muscle growth. We used our previously published mRNA (GSE72424) and miRNA (GSE62971) deep sequencing data from two-tailed samples [i.e., the highest (h) and lowest (l) body weights] of Recessive White Rock (WRR) and Xinghua (XH) chickens to conduct integrative analyses of the miRNA-mRNA interactions involved in chicken skeletal muscle growth. A total of 162, 15, 173, and 27 miRNA-mRNA pairs with negatively correlated expression patterns were identified in miRNA-mRNA networks constructed on the basis of the WRRh vs. XHh, WRRh vs. WRRl, WRRl vs. XHl, and XHh vs. XHl comparisons, respectively. Ingenuity Pathway Analysis revealed that gene networks identified for the WRRh vs. XHh contrast were associated with developmental disorders. Importantly, the WRRh vs. XHh contrast miRNA-mRNA network was enriched in IGF-1 signaling pathway genes, including FOXO3. A dual-luciferase reporter assay showed that FOXO3 was a target of miR-142-5p. Furthermore, miR-142-5p overexpression significantly decreased FOXO3 mRNA levels and promoted the expression of growth-related genes. These data demonstrated that miR-142-5p targets FOXO3 and promotes growth-related gene expression and regulates skeletal muscle growth in chicken. Comprehensive analysis facilitated the identification of miRNAs and target genes that might contribute to the regulation of skeletal muscle development. Our results provide new clues for understanding the molecular basis of chicken growth.

LncIRS1 controls muscle atrophy via sponging miR-15 family to activate IGF1-PI3K/AKT pathway.

BACKGROUND: Recent studies indicate important roles for long noncoding RNAs (lncRNAs) in the regulation of gene expression by acting as competing endogenous RNAs (ceRNAs). However, the specific role of lncRNAs in skeletal muscle atrophy is still unclear. Our study aimed to identify the function of lncRNAs that control skeletal muscle myogenesis and atrophy. METHODS: RNA sequencing was performed to identify the skeletal muscle transcriptome (lncRNA and messenger RNA) between hypertrophic broilers and leaner broilers. To study the 'sponge' function of lncRNA, we constructed a lncRNA-microRNA (miRNA)-gene interaction network by integrated our previous submitted skeletal muscle miRNA sequencing data. The primary myoblast cells and animal model were used to assess the biological function of the lncIRS1 in vitro or in vivo. RESULTS: We constructed a myogenesis-associated lncRNA-miRNA-gene network and identified a novel ceRNA lncRNA named lncIRS1 that is specifically enriched in skeletal muscle. LncIRS1 could regulate myoblast proliferation and differentiation in vitro, and muscle mass and mean muscle fibre in vivo. LncIRS1 increases gradually during myogenic differentiation. Mechanistically, lncIRS1 acts as a ceRNA for miR-15a, miR-15b-5p, and miR-15c-5p to regulate IRS1 expression, which is the downstream of the IGF1 receptor. Overexpression of lncIRS1 not only increased the protein abundance of IRS1 but also promoted phosphorylation level of AKT (p-AKT) a central component of insulin-like growth factor-1 pathway. Furthermore, lncIRS1 regulates the expression of atrophy-related genes and can rescue muscle atrophy. CONCLUSIONS: The newly identified lncIRS1 acts as a sponge for miR-15 family to regulate IRS1 expression, resulting in promoting skeletal muscle myogenesis and controlling atrophy.

LncRNA-Six1 Encodes a Micropeptide to Activate Six1 in Cis and Is Involved in Cell Proliferation and Muscle Growth.

Long non-coding RNAs (lncRNAs) play important roles in epigenetic regulation of skeletal muscle development. In our previous RNA-seq study (accession number GSE58755), we found that lncRNA-Six1 is an lncRNA that is differentially expressed between White Recessive Rock (WRR) and Xinghua (XH) chicken. In this study, we have further demonstrated that lncRNA-Six1 is located 432 bp upstream of the gene encoding the protein Six homeobox 1 (Six1). A dual-luciferase reporter assay identified that lncRNA-Six1 overlaps the Six1 proximal promoter. In lncRNA-Six1, a micropeptide of about 7.26 kDa was found to play an important role in the lncRNA-Six1 in cis activity. Overexpression of lncRNA-Six1 promoted the mRNA and protein expression level of the Six1 gene, while knockdown of lncRNA-Six1 inhibited Six1 expression. Moreover, tissue expression profiles showed that both the lncRNA-Six1 and the Six1 mRNA were highly expressed in chicken breast tissue. LncRNA-Six1 overexpression promoted cell proliferation and induced cell division. Conversely, its loss of function inhibited cell proliferation and reduced cell viability. Similar effects were observed after overexpression or knockdown of the Six1 gene. In addition, overexpression or knockdown of Six1 promoted or inhibited, respectively, the expression levels of muscle-growth-related genes, such as MYOG, MYHC, MYOD, IGF1R, and INSR. Taken together, these data demonstrate that lncRNA-Six1 carries out cis-acting regulation of the protein-encoding Six1 gene, and encodes a micropeptide to activate Six1 gene, thus promoting cell proliferation and being involved in muscle growth.

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.

Long non-coding RNAs (lncRNAs) play important roles in transcriptional and post-transcriptional regulation. However, little is currently known about the mechanisms by which they regulate skeletal muscle development in the chicken. In this study, we used RNA sequencing to profile the leg muscle transcriptome (lncRNA and mRNA) at three stages of skeletal muscle development in the chicken: embryonic day 11 (E11), embryonic day 16 (E16), and 1 day after hatching (D1). In total, 129, 132, and 45 differentially expressed lncRNAs, and 1798, 3072, and 1211 differentially expressed mRNAs were identified in comparisons of E11 vs. E16, E11 vs. D1, and E16 vs. D1, respectively. Moreover, we identified the cis- and trans-regulatory target genes of differentially expressed lncRNAs, and constructed lncRNA-gene interaction networks. In total, 126 and 200 cis-targets, and two and three trans-targets were involved in lncRNA-gene interaction networks that were constructed based on the E11 vs. E16, and E11 vs. D1 comparisons, respectively. The comparison of the E16 vs. D1 lncRNA-gene network comprised 25 cis-targets. We determined that lncRNA target genes are potentially involved in cellular development, and cellular growth and proliferation using Ingenuity Pathway Analysis. The gene networks identified for the E11 vs. D1 comparison were involved in embryonic development, organismal development and tissue development. The present study provides an RNA sequencing based evaluation of lncRNA function during skeletal muscle development in the chicken. Comprehensive analysis facilitated the identification of lncRNAs and target genes that might contribute to the regulation of different stages of skeletal muscle development.