RNA-Seq Reveals miRNA Role Shifts in Seven Stages of Skeletal Muscles in Goat Fetuses and Kids.

MicroRNAs (miRNAs) are indispensable for the regulation of skeletal muscle. We performed RNA sequencing (RNA-seq) to establish a comprehensive miRNA profiling of goats in seven stages, namely, 45- (F45), 65- (F65), 90- (F90), 120- (F120), and 135-day (F135) fetuses, newborn (B1), and 90-day-old (B90) kids. In total, 421 known miRNAs and 228 goat novel miRNAs were identified in the data, and the average abundance of 19 miRNAs in seven stages exceeds 10,000 reads per million. Furthermore, 420 differentially expressed miRNAs (DEmiRNAs) were identified in all comparison group at seven stages, 80 of which were uniquely differentially expressed in the B1 and B90 comparison groups. Pathway analysis indicated that this group was associated with the release of muscle hypertrophy and regulation of myoblast proliferation. Besides, 305 DEmiRNAs were clustered into three significantly enriched profiles (profiles 11, 16, and 19). Function analysis revealed that profile 16 was related to muscle hypertrophy and differentiation. Profile 11 was involved in multiple enzyme activities and metabolic processes in muscle cells. And profile 19 was involved in material transport and structural stability. Two highly expressed miRNAs and three key miRNAs (chi-miR-328-3p, chi-miR-767, and chi-miR-150) of these profiles were verified to be consistent with the data by quantitative real-time PCR. These results provided a catalog of goat muscle-associated miRNAs, allowing us to better understand the transformation of miRNA roles during mammalian muscle development.

Trend analysis of the role of circular RNA in goat skeletal muscle development.

BACKGROUND: Circular RNA (circRNA) is produced during the splicing of mRNA (in addition to linear splicing) and is part of the gene regulatory network. The temporal expression patterns the different developmental stages were inseparable from these molecules' function. RESULTS: Skeletal muscles of Anhui white goat (AWG) across seven fetal to postnatal development stages were sequenced and 21 RNA sequencing libraries were constructed. We thereby identified 9090 circRNAs and analyzed their molecular properties, temporal expression patterns, and potential functions at the different stages. CircRNAs showed complexities and diversity of formation as the same host gene produces multiple isoforms of these nucleic acids with different expression profiles. The differential expression of 2881 circRNAs (DECs, P < 0.05) was identified and four were randomly selected and validated by qPCR. Moreover, 1118 DECs under strict selected (SDECs, |log2FC| > 2 and P-adj value < 0.01) showed 4 expression trends (Clusters 0, 19, 16 and 18). Cluster 0 molecules had increasing expression at all stages with effects on muscle through metabolism, regulation of enzyme activity, and biosynthesis. Cluster 16 circRNAs had high expression in the early and late stages and are involved in "Wnt signaling pathway", "AMPK signaling pathway" and others. Cluster 18 molecules were mainly expressed at F120 and participate in "cytoskeletal protein binding", "Notch signaling pathway" and so on. Cluster 19 circRNAs were down-regulated at all stages and related to muscle structure and development. Lastly, the SDECs divided the period of skeletal muscle development into three transitional stages: stage 1 (F45 to F90), which related to muscle satellite cell proliferation and muscle fiber structure; stage 2 (F90 to B1), in which the attachment of the cytoplasmic surface to the actin cytoskeleton initiates; and stage 3, which involved the "cGMP-PKG signaling pathway". Moreover, the paraffin sections messages also validated that there are three transitional stages of skeletal muscle development. CONCLUSION: Our current study provides a catalog of goat muscle-related circRNAs that can stratify skeletal muscle development fetus 45 days to newborn 90 days into three developmental stages. These findings better our understanding of functional transitions during mammalian muscle development.

The expression and regulation of miR-1 in goat skeletal muscle and satellite cell during muscle growth and development.

MicroRNA-1 (miR-1) has been shown to play an important role in muscle growth and development, however, it was mainly discovered in model animals. To explore the function and mechanism of miR-1 in goat, we firstly explored the expression profile of miR-1 in goat tissues and cells. Furthermore, the target gene of miR-1 was predicted, and the relationship between miR-1 and one of its target genes, histone deacetylase 4 (HDAC4), was analyzed through double luciferase reporter assay, real-time PCR, and western blot. It was found that the miR-1 is most abundantly expressed in goat heart and skeletal muscle tissue. Meanwhile, the expression of miR-1 showed an increasing tendency from new-born goats to the 7-month-old goats, and then its expression decreases as the goats mature further. In addition, the expression levels of miR-1 decreased in goat skeletal muscle satellite cells with the algebraic increasing of cells. At last, the results showed that HDAC4 is a target gene of miR-1 in goat, and miR-1 can inhibit the post-transcriptional expression of HDAC4, but had no significant influence on the mRNA level of HDAC4. It was hypothesized that miR-1 promotes muscle development by inhibiting the post-transcriptional expression of HDAC4 in goat.

Identification of lncRNAs by RNA Sequencing Analysis During in Vivo Pre-Implantation Developmental Transformation in the Goat.

Pre-implantation development is a dynamic, complex and precisely regulated process that is critical for mammalian development. There is currently no description of the role of the long noncoding RNAs (lncRNAs) during the pre-implantation stages in the goat. The in vivo transcriptomes of oocytes (n = 3) and pre-implantation stages (n=19) at seven developmental stages in the goat were analyzed by RNA sequencing (RNA-Seq). The major zygotic gene activation (ZGA) event was found to occur between the 8- and 16-cell stages in the pre-implantation stages. We identified 5,160 differentially expressed lncRNAs (DELs) in developmental stage comparisons and functional analyses of the major and minor ZGAs. Fourteen lncRNA modules were found corresponding to specific pre-implantation developmental stages by weighted gene co-expression network analysis (WGCNA). A comprehensive analysis of the lncRNAs at each developmental transition of high correlation modules was done. We also identified lncRNA-mRNA networks and hub-lncRNAs for the high correlation modules at each stage. The extensive association of lncRNA target genes with other embryonic genes suggests an important regulatory role for lncRNAs in embryonic development. These data will facilitate further exploration of the role of lncRNAs in the developmental transformation in the pre-implantation stage.

Comprehensive Analysis of LncRNA Reveals the Temporal-Specific Module of Goat Skeletal Muscle Development

A series of complex processes regulate muscle development, and lncRNAs play essential roles in the regulation of skeletal myogenesis. Using RNA sequencing, we profiled the lncRNA expression during goat (Capra hircus) skeletal muscle development, which included seven stages across fetal 45 (F45), 65 (F65), 90 (F90), 120 (F120), 135 (F135) days, born for 24 h (B1) and 90 (B90) days. A total of 15,079 lncRNAs were identified in the seven stages, and they were less conservative with other species (human, cow, and mouse). Among them, 547 were differentially expressed, and they divided the seven stages into three functional transition periods. Following weighted gene co-expression network analysis (WGCNA), five lncRNA modules specific for developmental stages were defined as three types: 'Early modules', 'late modules', and 'individual-stage-specific modules'. The enrichment content showed that 'early modules' were related to muscle structure formation, 'late modules' participated in the 'p53 signaling pathway' and other pathways, the F90-highly related module was involved in the 'MAPK signaling pathway', and other pathways. Furthermore, we identified hub-lncRNA in three types of modules, and LNC_011371, LNC_ 007561, and LNC_001728 may play important roles in goat skeletal muscle. These data will facilitate further exploration of skeletal muscle lncRNA functions at different developmental stages in goats.

miR-27b regulates myogenic proliferation and differentiation by targeting Pax3 in goat.

This study found that miR-27 is expressed in muscle and regulates muscle proliferation and differentiation. We explored the function and regulatory mechanism of miR-27b in goat muscle proliferation and differentiation. Compared with the Boer goat, higher expression of miR-27b was observed in all of the collected muscle tissues of Anhuai goat, excluding the kidney, whereas the opposite expression pattern was observed for Pax3, which showed lower expression in Anhuai goat. Expression of miR-27b decreased gradually during the proliferation of skeletal muscle satellite cells in Anhuai goat and increased during differentiation; however, the expression pattern of Pax3 was opposite. The regulatory activity of miR-27b demonstrated that miR-27b inhibited the proliferation of skeletal muscle satellite cells, but promoted their differentiation. Moreover, function research demonstrated that Pax3 negatively regulated myogenic differentiation of goat skeletal muscle satellite cells, but accelerated their proliferation. The results of a dual-luciferase reporter analysis showed that miR-27b directly targeted the 3'-untranslated regions of Pax3 mRNA, and western blot and immunofluorescence staining analyses showed that miR-27b inhibited expression of the Pax3 protein. In goats, miR-27b can regulate myogenic proliferation and differentiation by targeting Pax3.

Identification and analysis of differentially expressed long non-coding RNAs between multiparous and uniparous goat (Capra hircus) ovaries.

Long non-coding RNAs (lncRNAs) play important roles in almost all biological processes. However, there is little information on the effects of lncRNAs on ovulation and lambing rates. In the present study, we used high-throughput RNA sequencing to identify differentially expressed lncRNAs between the ovaries of multiparous (Mul) and uniparous (Uni) Anhui White goats. Among the 107,255,422 clean reads, 183,754 lncRNAs were significantly differentially expressed between the Uni and Mul. Among them, 455 lncRNAs were co-expressed between the two samples, whereas, 157,523 lncRNAs were uniquely expressed in the Uni, and 25,776 uniquely lncRNAs were expressed in the Mul. Through Cis role analysis, 24 lncRNAs were predicted to overlap with cis-regulatory elements, which involved in Progesterone-mediated oocyte maturation, Steroid biosynthesis, Oocyte meiosis, and gonadotropin-releasing hormone (GnRH) signaling pathway. These 4 pathways were related to ovulation, and the KEGG pathway analysis on target genes of the differentially expressed lncRNAs confirmed this results. In addition, 10 lncRNAs harbored precursors of 40 miRNAs, such as TCONS_00320849 related to a mature miRNA sequence, miR-365a, which was reported to be related to proliferation, were annotated in the precursor analysis of miRNAs. The present expand the understanding of lncRNA biology and contribute to the annotation of the goat genome. The study will provide a resource for lncRNA studies of ovulation and lambing.

Comparative profiling of differentially expressed microRNAs between the follicular and luteal phases ovaries of goats.

To explore if the regulation at post-transcriptional level of follicular phase (Fols) to luteal phase (Luts) transition occurs in the ovaries of Anhuai goats, the differentially expressed microRNAs (miRNAs) of ovaries in the Fols and Luts were analyzed using Solexa sequencing in the study. In total, 320 known miRNAs were co-expressed in the two phases, 339 and 353 known miRNAs were expressed in the ovary in the Fols and Luts, respectively. In addition, 45 novel miRNAs were co-expressed in the two phases, 70 and 94 novel miRNAs were expressed in the ovary in the Fols and Luts, respectively. Let-7f was the highest expressed significantly different known miRNA in the two phases, and mir-159 was the highest expressed significantly different novel miRNA in the two phases, which may participate in the follicular-luteal transition of Anhuai goats. GO annotation and KEGG pathway analysis were applied to analyze the target genes of differentially expressed miRNAs detected in the two phases. The results will help to further understand the role of miRNAs in the regulation of follicular to luteal transition in goat ovaries.