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 WRR vs. XH, WRR vs. WRR, WRR vs. XH, and XH vs. XH comparisons, respectively. Ingenuity Pathway Analysis revealed that gene networks identified for the WRR vs. XH contrast were associated with developmental disorders. Importantly, the WRR vs. XH 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.

Li Z ，Abdalla BA ，Zheng M ，He X ，Cai B ，Han P ，Ouyang H ，Chen B ，Nie Q ，Zhang X ... -  《-》

Deep Sequencing Analysis of miRNA Expression in Breast Muscle of Fast-Growing and Slow-Growing Broilers.

Growth performance is an important economic trait in chicken. MicroRNAs (miRNAs) have been shown to play important roles in various biological processes, but their functions in chicken growth are not yet clear. To investigate the function of miRNAs in chicken growth, breast muscle tissues of the two-tail samples (highest and lowest body weight) from Recessive White Rock (WRR) and Xinghua Chickens (XH) were performed on high throughput small RNA deep sequencing. In this study, a total of 921 miRNAs were identified, including 733 known mature miRNAs and 188 novel miRNAs. There were 200, 279, 257 and 297 differentially expressed miRNAs in the comparisons of WRRh vs. WRRl, WRRh vs. XHh, WRRl vs. XHl, and XHh vs. XHl group, respectively. A total of 22 highly differentially expressed miRNAs (fold change > 2 or < 0.5; p-value < 0.05; q-value < 0.01), which also have abundant expression (read counts > 1000) were found in our comparisons. As far as two analyses (WRRh vs. WRRl, and XHh vs. XHl) are concerned, we found 80 common differentially expressed miRNAs, while 110 miRNAs were found in WRRh vs. XHh and WRRl vs. XHl. Furthermore, 26 common miRNAs were identified among all four comparisons. Four differentially expressed miRNAs (miR-223, miR-16, miR-205a and miR-222b-5p) were validated by quantitative real-time RT-PCR (qRT-PCR). Regulatory networks of interactions among miRNAs and their targets were constructed using integrative miRNA target-prediction and network-analysis. Growth hormone receptor (GHR) was confirmed as a target of miR-146b-3p by dual-luciferase assay and qPCR, indicating that miR-34c, miR-223, miR-146b-3p, miR-21 and miR-205a are key growth-related target genes in the network. These miRNAs are proposed as candidate miRNAs for future studies concerning miRNA-target function on regulation of chicken growth.

Ouyang H ，He X ，Li G ，Xu H ，Jia X ，Nie Q ，Zhang X ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

A Genome-Wide mRNA Screen and Functional Analysis Reveal FOXO3 as a Candidate Gene for Chicken Growth.

Chicken growth performance provides direct economic benefits to the poultry industry. However, the underlying genetic mechanisms are unclear. The objective of this study was to identify candidate genes associated with chicken growth and investigate their potential mechanisms. We used RNA-Seq to study the breast muscle transcriptome in high and low tails of Recessive White Rock (WRRh, WRRl) and Xinghua chickens (XHh, XHl). A total of 60, 23, 153 and 359 differentially expressed genes were detected in WRRh vs. WRRl, XHh vs. XHl, WRRh vs. XHh and WRRl vs. XHl, respectively. GO, KEGG pathway and gene network analyses showed that CEBPB, FBXO32, FOXO3 and MYOD1 played key roles in growth. The functions of FBXO32 and FOXO3 were validated. FBXO32 was predominantly expressed in leg muscle, heart and breast muscle. After decreased FBXO32 expression, growth-related genes such as PDK4, IGF2R and IGF2BP3 were significantly down-regulated (P < 0.05). FBXO32 was significantly (P < 0.05) associated with carcass and meat quality traits, but not growth traits. FOXO3 was predominantly expressed in breast and leg muscle. In both of these tissues, the FOXO3 mRNA level in XH was significantly higher than that in WRR chickens with normal body weight (P < 0.05). In DF-1 cells, siRNA knockdown of FOXO3 significantly (P < 0.01) inhibited the MYOD expression and significantly up-regulated (P < 0.01 or P < 0.05) the expression of growth-related genes including CEBPB, FBXO32, GH, GHR, IGF1R, IGF2R, IGF2BP1, IGF2BP3, INSR, PDK1 and PDK4. Moreover, 18 SNPs were identified in FOXO3. G66716193A was significantly (P < 0.05) associated with growth traits. The sites C66716002T, C66716195T and A66716179G were significantly (P < 0.05) associated with growth or carcass traits. These results demonstrated that FOXO3 is a candidate gene influencing chicken growth. Our observations provide new clues to understand the molecular basis of chicken growth.

Chen B ，Xu J ，He X ，Xu H ，Li G ，Du H ，Nie Q ，Zhang X ... -  《PLoS One》

MicroRNA profiling associated with muscle growth in modern broilers compared to an unselected chicken breed.

Genetically selected modern broiler chickens have acquired outstanding production efficiency through rapid growth and improved feed efficiency compared to unselected chicken breeds. Recently, we analyzed the transcriptome of breast muscle tissues obtained from modern pedigree male (PeM) broilers (rapid growth and higher efficiency) and foundational Barred Plymouth Rock (BPR) chickens (slow growth and poorer efficiency). This study was designed to investigate microRNAs that play role in rapid growth of the breast muscles in modern broiler chickens. In this study, differential abundance of microRNA (miRNA) was analyzed in breast muscle of PeM and BPR chickens and the results were integrated with differentially expressed (DE) mRNA in the same tissues. A total of 994 miRNA were identified in PeM and BPR chicken lines from the initial analysis of small RNA sequencing data. After filtering and statistical analyses, the results showed miR-2131-5p, miR-221-5p, miR-126-3p, miR-146b-5p, miR-10a-5p, let-7b, miR-125b-5p, and miR-146c-5p up-regulated whereas miR-206 down-regulated in PeM compared to BPR breast muscle. Based on inhibitory regulations of miRNAs on the mRNA abundance, our computational analysis using miRDB, an online software, predicated that 118 down-regulated mRNAs may be targeted by the up-regulated miRNAs, while 35 up-regulated mRNAs appear to be due to a down-regulated miRNA (i.e., miR-206). Functional network analyses of target genes of DE miRNAs showed their involvement in calcium signaling, axonal guidance signaling, and NRF2-mediated oxidative stress response pathways suggesting their involvement in breast muscle growth in chickens. From the integrated analyses of differentially expressed miRNA-mRNA data, we were able to identify breast muscle specific miRNAs and their target genes whose concerted actions can contribute to rapid growth and higher feed efficiency in modern broiler chickens. This study provides foundation data for elucidating molecular mechanisms that govern muscle growth in chickens.

Khatri B ，Seo D ，Shouse S ，Pan JH ，Hudson NJ ，Kim JK ，Bottje W ，Kong BC ... -  《BMC GENOMICS》

Altered miRNA and mRNA Expression in Sika Deer Skeletal Muscle with Age.

Studies of the gene and miRNA expression profiles associated with the postnatal late growth, development, and aging of skeletal muscle are lacking in sika deer. To understand the molecular mechanisms of the growth and development of sika deer skeletal muscle, we used de novo RNA sequencing (RNA-seq) and microRNA sequencing (miRNA-seq) analyses to determine the differentially expressed (DE) unigenes and miRNAs from skeletal muscle tissues at 1, 3, 5, and 10 years in sika deer. A total of 51,716 unigenes, 171 known miRNAs, and 60 novel miRNAs were identified based on four mRNA and small RNA libraries. A total of 2,044 unigenes and 11 miRNAs were differentially expressed between adolescence and juvenile sika deer, 1,946 unigenes and 4 miRNAs were differentially expressed between adult and adolescent sika deer, and 2,209 unigenes and 1 miRNAs were differentially expressed between aged and adult sika deer. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that DE unigenes and miRNA were mainly related to energy and substance metabolism, processes that are closely associate with the growth, development, and aging of skeletal muscle. We also constructed mRNA-mRNA and miRNA-mRNA interaction networks related to the growth, development, and aging of skeletal muscle. The results show that mRNA (Myh1, Myh2, Myh7, ACTN3, etc.) and miRNAs (miR-133a, miR-133c, miR-192, miR-151-3p, etc.) may play important roles in muscle growth and development, and mRNA (WWP1, DEK, UCP3, FUS, etc.) and miRNAs (miR-17-5p, miR-378b, miR-199a-5p, miR-7, etc.) may have key roles in muscle aging. In this study, we determined the dynamic miRNA and unigenes transcriptome in muscle tissue for the first time in sika deer. The age-dependent miRNAs and unigenes identified will offer insights into the molecular mechanism underlying muscle development, growth, and maintenance and will also provide valuable information for sika deer genetic breeding.

Jia B ，Liu Y ，Li Q ，Zhang J ，Ge C ，Wang G ，Chen G ，Liu D ，Yang F ... -  《Genes》