MiR-208b regulates cell cycle and promotes skeletal muscle cell proliferation by targeting CDKN1A.

Skeletal muscle is the most abundant tissue in the body. The development of skeletal muscle cell is complex and affected by many factors. A sea of microRNAs (miRNAs) have been identified as critical regulators of myogenesis. MiR-208b, a muscle-specific miRNA, was reported to have a connection with fiber type determination. However, whether miR-208b has effect on proliferation of muscle cell was under ascertained. In our study, cyclin-dependent kinase inhibitor 1A (CDKN1A), which participates in cell cycle regulation, was predicted and then validated as one target gene of miR-208b. We found that overexpression of miR-208b increased the expression of cyclin D1, cyclin E1, and cyclin-dependent kinase 2 at the levels of messenger RNA and protein in cattle primary myoblasts in vivo and in vitro. Flow cytometry showed that forced expression of miR-208b increased the percentage of cells at the S phase and decreased the percentage of cells at the G0/G1 phase. These results indicated that miR-208b participates in the cell cycle regulation of cattle primary myoblast cells. 5-Ethynyl-20-deoxyuridine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that overexpression of miR-208b promoted the proliferation of cattle primary myoblasts. Therefore, we conclude that miR-208b participates in the cell cycle and proliferation regulation of cattle primary skeletal muscle cell through the posttranscriptional downregulation of CDKN1A.

Genetic Variants in ADD1 Gene and their Associations with Growth Traits in Cattle.

The α-adducin (ADD1) is a subunit of adducin which is a cytoskeleton heterodimeric protein. Adducin participates in oocytes chromosome meiosis of mice, prompting adducin has an effect on embryonic development. Adducin gene mutation has significantly functional change. So the present study was to identify and characterize polymorphisms within the coding region of the bovine ADD1 gene among different cattle breeds. Here, 11 novel single nucleotide polymorphisms (SNPs 1-11) were identified by DNA sequencing and polymerase chain reaction-single stranded conformational polymorphism, there were one synonymous mutation in exon 1 (SNP1); four missense mutations in exons 4, 7, and 8 (SNPs 3-6); and six mutations in introns 4, 12, 13, and 14 (SNPs 2, 7-10). The statistical analyses indicated that the some SNPs are associated with the growth traits (body length, body height, chest circumference, and hucklebone width) in Chinese Jiaxian cattle population. Our results provide evidence that polymorphisms in the ADD1 gene are associated with growth traits, and may be used for marker-assisted selection in beef cattle breeding program.

Over-expression of DEC1 inhibits myogenic differentiation by modulating MyoG activity in bovine satellite cell.

Differentiated embryo chondrocyte 1 (DEC1), a member of basic-helix-loop-helix transcription factor Bhlhe40, also called stimulated by retinoic acid 13, STRA13, plays an important role in the regulation of adipogenesis, tumorigenesis, peripheral circadian output, response to hypoxia, and development of metabolic syndrome. Previous studies suggested that DEC1 was involved in skeletal muscle development; however, its precise role in myoblast differentiation has not been determined. In the present study, we showed that DEC1 expressed ubiquitously in different bovine tissues and was down-regulated in differentiated bovine satellite cells. Expression of muscle specific transcription factors (Myf5, MyoD, MyoG, and MHC) was significantly down-regulated when DEC1 was over-expressed by both CoCl2 -simulated hypoxia and Adenovirus-mediated transduction in bovine satellite cells. Consistent with that, promoter analyses via luciferase reporter assay also revealed that overexpression of bovine DEC1 could inhibit MyoG promoter activity. In conclusion, overexpression of DEC1 blocked myogenesis by inhibiting MyoG promoter activity in bovine. Our results provided a new mechanism for the muscle growth, which would contribute to increase cattle meat productivity.

Integrating CNVs into meta-QTL identified GBP4 as positional candidate for adult cattle stature.

Copy number variation (CNV) of DNA sequences, functionally significant but yet fully ascertained, is believed to confer considerable increments in unexplained heritability of quantitative traits. Identification of phenotype-associated CNVs (paCNVs) therefore is a pressing need in CNV studies to speed up their exploitation in cattle breeding programs. Here, we provided a new avenue to achieve this goal that is to project the published CNV data onto meta-quantitative trait loci (meta-QTL) map which connects causal genes with phenotypes. Any CNVs overlapping meta-QTL therefore will be potential paCNVs. This study reported potential paCNVs in Bos taurus autosome 3 (BTA3). Notably, overview indexes and CNVs both highlighted a narrower region (BTA3 54,500,000-55,000,000 bp, named BTA3_INQTL_6) within one constructed meta-QTL. Then, we ascertained guanylate-binding protein 4 (GBP4) among the nine positional candidate genes was significantly associated with adult cattle stature, including body weight (BW, P < 0.05) and withers height (WHT, P < 0.05), fitting GBP4 CNV either with three levels or with six levels in the model. Although higher copy number downregulated the mRNA levels of GBP2 (P < 0.05) and GBP4 (P < 0.05) in 1-Mb window (54.0-55.0 Mb) in muscle and adipose, additional analyses will be needed to clarify the causality behind the ascertained association.

Long non-coding RNA ADNCR suppresses adipogenic differentiation by targeting miR-204.

Adipogenesis is a complex and precisely orchestrated process mediated by a network of adipogenic regulatory factors. Several studies have highlighted the relevance of lncRNAs in adipocyte differentiation, but the precise molecular mechanism has largely remained elusive. In the present study, we performed Ribo-Zero RNA-Seq to investigate both the poly(A)+and poly(A)-lncRNAs of in vitro cultured bovine preadipocytes and differentiated adipocytes. A stringent set of 2882 lncRNAs was finally identified. A comparison of the lncRNAs expression profiles revealed that 16 lncRNAs are differentially expressed during adipocyte differentiation. We focused on the most downregulated lncRNA, which we named adipocyte differentiation-associated long noncoding RNA (ADNCR). Mechanistically, ADNCR inhibited adipocyte differentiation by functioning as a competing endogenous RNA (ceRNA) for miR-204, thereby augmenting the expression of the miR-204 target gene, SIRT1, which is known to inhibit adipocyte differentiation and adipogenic gene expression by docking with NCoR and SMART to repress PPARγ activity. Our data not only provide a valuable genomic resource for the identification of lncRNAs with functional roles in adipocyte differentiation but also reveal new insights into understanding the mechanisms of adipogenic differentiation.

miR-378a-3p promotes differentiation and inhibits proliferation of myoblasts by targeting HDAC4 in skeletal muscle development.

Muscle development, or myogenesis, is a highly regulated, complex process. A subset of microRNAs (miRNAs) have been identified as critical regulators of myogenesis. Recently, miR-378a was found to be involved in myogenesis, but the mechanism of how miR-378a regulates the proliferation and differentiation of myoblasts has not been determined. We found that miR-378a-3p expression in muscle was significantly higher than in other tissues, suggesting an important effect on muscle development. Overexpression of miR-378a-3p increased the expression of MyoD and MHC in C2C12 myoblasts both at the level of mRNA and protein, confirming that miR-378a-3p promoted muscle cell differentiation. The forced expression of miR-378a-3p promoted apoptosis of C2C12 cells as evidenced by CCK-8 assay and Annexin V-FITC/PI staining results. Through TargetScan, histone acetylation enzyme 4 (HDAC4) was identified as a potential target of miR-378a-3p. We confirmed targeting of HDAC4 by miR-378a-3p using a dual luciferase assay and western blotting. Our RNAi analysis results also showed that HDAC4 significantly promoted differentiation of C2C12 cells and inhibited cell survival through Bcl-2. Therefore, we conclude that miR-378a-3p regulates skeletal muscle growth and promotes the differentiation of myoblasts through the post-transcriptional down-regulation of HDAC4.

miR-30-5p Regulates Muscle Differentiation and Alternative Splicing of Muscle-Related Genes by Targeting MBNL.

MicroRNAs (miRNAs), a class of single stranded, small (~22 nucleotides), non-coding RNAs, play an important role in muscle development. We focused on the role of the miR-30-5p family during bovine muscle development from previous high-throughput sequencing results and analyzed their expression profiles. MHC and MyoG mRNAs expression as well as their proteins were suppressed in differentiated C2C12 cells, suggesting the importance of miR-30-5p in muscle development. MBNL, the candidate target of miR-30-5p, is an alternative splicing regulation factor. MBNL1 and MBNL3 have opposite effects on muscle differentiation. Our results confirmed that miR-30a-5p and miR-30e-5p repress the expression of MBNL1, MBNL2 and MBNL3, whereas miR-30b-5p inhibits MBNL1 and MBNL2 expression. This provides direct evidence that MBNL expression can be flexibly regulated by miR-30-5p. Previous studies showed that MBNL1 promotes exon inclusion of two muscle-related genes (Trim55 and INSR). Through RNA splicing studies, we found that miR-30-5p had an effect on their alternative splicing, which means miR-30-5p via MBNL1 could be integrated into muscle signaling pathways in which INSR or Trim55 are located. In conclusion, miR-30-5p could inhibit muscle cell differentiation and regulate the alternative splicing of Trim55 and INSR by targeting MBNL. These results promote the understanding of the function of miRNAs in muscle development.

Global Transcriptome Analysis During Adipogenic Differentiation and Involvement of Transthyretin Gene in Adipogenesis in Cattle.

Adipose tissue plays central role in determining the gustatory quality of beef, but traditional Chinese beef cattle have low levels of fat content. We applied RNA-seq to study the molecular mechanisms underlying adipocyte differentiation in Qinchuan cattle. A total of 18,283 genes were found to be expressed in preadipocytes and mature adipocytes, respectively. 470 of which were significantly differentially expressed genes (DEGs) [false discovery rate (FDR) values < 0.05 and fold change ≥ 2]. In addition, 4534 alternative splicing (AS) events and 5153 AS events were detected in preadipocytes and adipocytes, respectively. We constructed a protein interaction network, which suggested that collagen plays an important role during bovine adipogenic differentiation. We characterized the function of the most down-regulated DEG (P < 0.001) among genes we have detected by qPCR, namely, the transthyretin (TTR) gene. Overexpression of TTR appears to promote the expression of the peroxisome proliferator activated receptor γ (PPARγ) (P < 0.05) and fatty acid binding Protein 4 (FABP4) (P < 0.05). Hence, TTR appears to be involved in the regulation of bovine adipogenic differentiation. Our study represents the comprehensive approach to explore bovine adipocyte differentiation using transcriptomic data and reports an involvement of TTR during bovine adipogenic differentiation. Our results provide novel insights into the molecular mechanisms underlying bovine adipogenic differentiation.

circFGFR4 Promotes Differentiation of Myoblasts via Binding miR-107 to Relieve Its Inhibition of Wnt3a.

Muscle development is regulated under a series of complicate processes, and non-coding RNAs, such as microRNAs (miRNAs) and circular RNAs (circRNAs), have been reported to play important roles in regulating myoblast proliferation and differentiation. We found that miR-107 expression was high in skeletal muscle of Qinchuan cattle. Overexpression of miR-107 inhibited bovine myoblasts differentiation and protected cells from apoptosis. Wnt3a was identified as a target of miR-107 by luciferase activity, real-time qPCR, and western blotting assays. Knockdown of Wnt3a inhibited bovine myoblasts differentiation and apoptosis, and this effect was similar to miR-107 overexpression. We also found circFGFR4 to promote myoblasts differentiation and to induce cell apoptosis. Via luciferase screening and RNA pull-down assays, circFGFR4 was observed to sponge miR-107. Overexpression of circFGFR4 increased the expression of Wnt3a, whereas this effect was abolished by miR-107. These results demonstrated that circFGFR4 binding miR-107 promotes cell differentiation via targeting Wnt3a in bovine primary myoblasts.

Circular RNA profiling reveals an abundant circLMO7 that regulates myoblasts differentiation and survival by sponging miR-378a-3p.

Circular RNAs (circRNAs) have been identified from various tissues and species, but their regulatory functions during developmental processes are not well understood. We examined circRNA expression profiles of two developmental stages of bovine skeletal muscle (embryonic and adult musculus longissimus) to provide first insights into their potential involvement in bovine myogenesis. We identified 12 981 circRNAs and annotated them to the Bos taurus reference genome, including 530 circular intronic RNAs (ciRNAs). One parental gene could generate multiple circRNA isoforms, with only one or two isoforms being expressed at higher expression levels. Also, several host genes produced different isoforms when comparing development stages. Most circRNA candidates contained two to seven exons, and genomic distances to back-splicing sites were usually less than 50 kb. The length of upstream or downstream flanking introns was usually less than 105 nt (mean≈11 000 nt). Several circRNAs differed in abundance between developmental stages, and real-time quantitative PCR (qPCR) analysis largely confirmed differential expression of the 17 circRNAs included in this analysis. The second part of our study characterized the role of circLMO7-one of the most down-regulated circRNAs when comparing adult to embryonic muscle tissue-in bovine muscle development. Overexpression of circLMO7 inhibited the differentiation of primary bovine myoblasts, and it appears to function as a competing endogenous RNA for miR-378a-3p, whose involvement in bovine muscle development has been characterized beforehand. Congruent with our interpretation, circLMO7 increased the number of myoblasts in the S-phase of the cell cycle and decreased the proportion of cells in the G0/G1 phase. Moreover, it promoted the proliferation of myoblasts and protected them from apoptosis. Our study provides novel insights into the regulatory mechanisms underlying skeletal muscle development and identifies a number of circRNAs whose regulatory potential will need to be explored in the future.

Application of mathematical expectation (ME) strategy for detecting low frequency mutations: An example for evaluating 14-bp insertion/deletion (indel) within the bovine PRNP gene.

The detection method based on the mathematical expectation (ME) strategy is fast and accuracy for low frequency mutation screening in large samples. Previous studies have found that the 14-bp insertion/deletion (indel) variants of the 3' untranslated region (3' UTR) within bovine PRNP gene have been characterized with low frequency (≤5%) in global breeds outside China, which has not been determined in Chinese cattle breeds yet. Therefore, this study aimed to identify the 14-bp indel within PRNP gene in 5 major Chinese indigenous cattle breeds and to evaluate its associations with phenotypic traits. It was the first time to use ME strategy to detect low frequency indel polymorphisms and found that minor allele frequency was 0.038 (Qinchuan), 0.033 (Xianan), 0.013 (Nanyang), 0.003 (Jiaxian), and zero (Ji'an), respectively. Compared to the traditional detection method by which the sample was screened one by one, the reaction time by using the ME method was decreased 62.5%, 64.9%, 77.6%, 88.9% and 66.4%, respectively. In addition, the 14-bp indel was significantly associated with the growth traits in 2 cattle breeds, with the body length of Qinchuan cattle as well as the body weight and waistline of Xianan cattle. Our results have uncovered that the method based on ME strategy is rapid, reliable, and cost-effective for detecting the low frequency mutation as well as our findings provide a potential valuable theoretical basis for the marker-assisted selection (MAS) in beef cattle.