Hanfang Cai

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-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.

The developmental transcriptome sequencing of bovine skeletal muscle reveals a long noncoding RNA, lncMD, promotes muscle differentiation by sponging miR-125b.

Pervasive transcription of the mammalian genome generates numerous long noncoding RNAs (lncRNAs), which are of crucial importance in diverse biological processes. Recent advances in high throughput sequencing technology have helped to accelerate the pace of lncRNA discovery. However, no study on the overall expression patterns of lncRNAs during muscle development has been conducted. We reported here the first analysis of lncRNA landscape in bovine embryonic, neonatal and adult skeletal muscle using Ribo-Zero RNA-Seq, a technology which can capture both poly(A)+ and poly(A)- transcripts. We finally defined 7692 high-confidence lncRNAs and uncovered 401 lncRNAs differentially expressed among three developmental stages, including lncMD, a novel muscle-specific lncRNA which is gradually up-regulated during myoblast differentiation. lncMD overexpression upregulated, whereas lncMD silencing decreased the expression of two well-established myogenic markers, myosin heavy chain (MHC) and myogenin (MyoG). In-depth analyses showed that lncMD acts as a molecular sponge for miR-125b and that insulin-like growth factor 2 (IGF2) is a direct target of miR-125b in cattle. Moreover, lncMD level was positively correlated with IGF2 mRNA level in bovine muscle tissues, a vital corollary to ceRNA function. Altogether, our research showed that lncMD acts as a ceRNA to sequester miR-125b, leading to heightened IGF2 expression and thus promotes muscle differentiation. Our findings also complement the reference genome annotation of cattle, which will likely be useful for further functional lncRNA cloning and more comprehensive studies on lncRNA regulation in muscle development.

Characterization of Transcriptional Complexity during Adipose Tissue Development in Bovines of Different Ages and Sexes

Background Adipose tissue has long been recognized to play an extremely important role in development. In bovines, it not only serves a fundamental function but also plays a key role in the quality of beef and, consequently, has drawn much public attention. Age and sex are two key factors that affect the development of adipose tissue, and there has not yet been a global study detailing the effects of these two factors on expressional differences of adipose tissues. Results In this study, total RNA from the back fat of fetal bovines, adult bulls, adult heifers and adult steers were used to construct libraries for Illumina next-generation sequencing. We detected the expression levels of 12,233 genes, with over 3,000 differently expressed genes when comparing fetal and adult patterns and an average of 1000 differently expressed genes when comparing adult patterns. Multiple Gene Ontology terms and pathways were found to be significantly enriched for these differentially expressed genes. Of the 12,233 detected genes, a total of 4,753 genes (38.85%) underwent alternative splicing events, and over 50% were specifically expressed in each library. Over 4,000 novel transcript units were discovered for one library, whereas only approximately 30% were considered to have coding ability, which supplied a large amount of information for the lncRNA study. Additionally, we detected 56,564 (fetal bovine), 65,154 (adult bull), 78,061 (adult heifer) and 86,965 (adult steer) putative single nucleotide polymorphisms located in coding regions of the four pooled libraries. Conclusion Here, we present, for the first time, a complete dataset involving the spatial and temporal transcriptome of bovine adipose tissue using RNA-seq. These data will facilitate the understanding of the effects of age and sex on the development of adipose tissue and supply essential information towards further studies on the genomes of beef cattle and other related mammals.

Associations of MYH3 gene copy number variations with transcriptional expression and growth traits in Chinese cattle

Copy number variations (CNVs) have been recently recognized as another important genetic variability complementary to single nucleotide polymorphisms (SNPs). Compelling evidence has indicated that CNVs are responsible for phenotypic traits by changing the copy numbers of functional genes. Myosin heavy chain 3 (MYH3) gene is a critical regulatory factor in skeletal muscle development, and has been detected in the CNVs region by comparative genomic hybridization (CGH) array. This study was conducted to validate and detect the distribution of MYH3 copy numbers (relative to Angus cattle) in four Chinese cattle breeds (NY, QC, LX, and CY), and further to investigate the associations of the copy number changes with its transcriptional expression and cattle growth traits. Substantial genetic differences of MYH3 copy numbers were identified between NY and the other three breeds (P<0.01). The copy numbers of MYH3 gene presented the positive correlations with the transcript level of MYH3 gene in both fetal and adult skeletal muscles (P<0.05). Statistical analysis revealed that CNVs of MYH3 gene were significantly associated with growth traits of NY cattle, and the individuals with copy number gain showed better phenotypes than the loss and/or median groups (P<0.05). This study firstly attempted to establish the correlations between CNVs of candidate genes and growth traits, and our results suggested that the CNVs of MYH3 gene may be utilized as the potential markers for economic traits in selection breeding programs of Chinese cattle.

The developmental transcriptome landscape of bovine skeletal muscle defined by Ribo-Zero ribonucleic acid sequencing.

Ribonucleic acid sequencing (RNA-Seq) libraries are normally prepared with oligo(dT) selection of poly(A)+ mRNA, but it depends on intact total RNA samples. Recent studies have described Ribo-Zero technology, a novel method that can capture both poly(A)+ and poly(A)- transcripts from intact or fragmented RNA samples. We report here the first application of Ribo-Zero RNA-Seq for the analysis of the bovine embryonic, neonatal, and adult skeletal muscle whole transcriptome at an unprecedented depth. Overall, 19,893 genes were found to be expressed, with a high correlation of expression levels between the calf and the adult. Hundreds of genes were found to be highly expressed in the embryo and decreased at least 10-fold after birth, indicating their potential roles in embryonic muscle development. In addition, we present for the first time the analysis of global transcript isoform discovery in bovine skeletal muscle and identified 36,694 transcript isoforms. Transcriptomic data were also analyzed to unravel sequence variations; 185,036 putative SNP and 12,428 putative short insertions-deletions (InDel) were detected. Specifically, many stop-gain, stop-loss, and frameshift mutations were identified that probably change the relative protein production and sequentially affect the gene function. Notably, the numbers of stage-specific transcripts, alternative splicing events, SNP, and InDel were greater in the embryo than in the calf and the adult, suggesting that gene expression is most active in the embryo. The resulting view of the transcriptome at a single-base resolution greatly enhances the comprehensive transcript catalog and uncovers the global trends in gene expression during bovine skeletal muscle development.

Discovery of novel and differentially expressed microRNAs between fetal and adult backfat in cattle.

The posttranscriptional gene regulation mediated by microRNAs (miRNAs) plays an important role in various species. Recently, a large number of miRNAs and their expression patterns have been identified. However, to date, limited miRNAs have been reported to modulate adipogenesis and lipid deposition in beef cattle. Total RNAs from Chinese Qinchuan bovine backfat at fetal and adult stages were used to construct small RNA libraries for Illumina next-generation sequencing. A total of 13,915,411 clean reads were obtained from a fetal library and 14,244,946 clean reads from an adult library. In total, 475 known and 36 novel miRNA candidates from backfat were identified. The nucleotide bias, base editing, and family of the known miRNAs were also analyzed. Based on stem-loop qPCR, 15 specific miRNAs were detected, and the results showed that bta-miRNAn25 and miRNAn26 were highly expressed in backfat tissue, suggesting these small RNAs play a role in the development and maintenance of bovine subcutaneous fat tissue. Putative targets for miRNAn25 and miRNAn26 were predicted, and the 61 most significant target transcripts were related to lipid and fatty acid metabolism. Of interest, the canonical pathway and gene networks analyses revealed that PPARα/RXRα activation and LXR/RXR activation were important components of the gene interaction hierarchy results. In the present study, we explored the backfat miRNAome differences between cattle of different developmental stages, expanding the expression repertoire of bovine miRNAs that could contribute to further studies on the fat development of cattle. Predication of target genes analysis of miRNA25 and miRNA26 also showed potential gene networks that affect lipid and fatty acid metabolism. These results may help in the design of new intervention strategies to improve beef quality.

Identification of novel alternative splicing transcript and expression analysis of bovine TMEM95 gene.

Transmembrane protein 95 (TMEM95) is closely related to male reproductive performance in cattle, but does not affect semen quality. Alternative splicing plays an important role in regulating biological function as well as in generating proteomic and functional diversity in metazoan organisms. Thus, the aim of this study was to clone and identify transcripts of the TMEM95 gene in cattle using RT-PCR, characterize them via bioinformatics analysis, and detect their expression patterns using qRT-PCR. Two transcripts of TMEM95 were identified in cattle, including TMEM95-SV1 and TMEM95-SV2. Bioinformatics predicted that TMEM95-SV1 has a leucine-rich repeat C-terminal domain and a Pfam: IZUMO. These regions are closely related to protein interactions and the acrosome reaction, respectively. Interestingly, the two transcripts were exclusively expressed in the testes and brain in male fetus cattle, and TMEM95-SV1 was expressed in the brain at significantly higher levels than in the testis (P<0.05, 4.06-fold) and TMEM95-SV2 in the brain (P<0.05, 4.95-fold). These findings enrich the understanding of the TMEM95 gene function and benefit for enhancing male reproduction in cattle industry.

SNPs of bovine HGF gene and their association with growth traits in Nanyang cattle.

Hepatocyte growth factor (HGF) is one of the multifunctional cell factors that regulates cellular proliferation, motility and morphogenesis in mammalians. And its medical research has deep significance. In this paper, polymorphisms of HGF gene were investigated in 1433 health and irrelated Chinese cattle by PCR-RFLP and DNA sequencing approach. Ten novel Single nucleotide polymorphisms (SNPs) were identified, which included one missense mutation, g.72801G>A in the coding region, and the others in the intron. Association analysis between four of them, g.288T>C, g.72801G>A, g.77172G>T, and g.77408T>G, and growth traits in Nanyang, were performed. The results indicated that SNPs within bovine HGF gene were significantly associated with growth traits. Phylogenetic analysis showed that the genetic background of Caoyuan Red cattle was different from the others in the tested breeds. The findings will provide a background for application of bovine HGF gene in the selection program in Chinese cattle.

Whole-genome sequencing reveals mutational landscape underlying phenotypic differences between two widespread Chinese cattle breeds

Whole-genome sequencing provides a powerful tool to obtain more genetic variability that could produce a range of benefits for cattle breeding industry. Nanyang (Bos indicus) and Qinchuan (Bos taurus) are two important Chinese indigenous cattle breeds with distinct phenotypes. To identify the genetic characteristics responsible for variation in phenotypes between the two breeds, in the present study, we for the first time sequenced the genomes of four Nanyang and four Qinchuan cattle with 10 to 12 fold on average of 97.86% and 98.98% coverage of genomes, respectively. Comparison with the Bos_taurus_UMD_3.1 reference assembly yielded 9,010,096 SNPs for Nanyang, and 6,965,062 for Qinchuan cattle, 51% and 29% of which were novel SNPs, respectively. A total of 154,934 and 115,032 small indels (1 to 3 bp) were found in the Nanyang and Qinchuan genomes, respectively. The SNP and indel distribution revealed that Nanyang showed a genetically high diversity as compared to Qinchuan cattle. Furthermore, a total of 2,907 putative cases of copy number variation (CNV) were identified by aligning Nanyang to Qinchuan genome, 783 of which (27%) encompassed the coding regions of 495 functional genes. The gene ontology (GO) analysis revealed that many CNV genes were enriched in the immune system and environment adaptability. Among several CNV genes related to lipid transport and fat metabolism, Lepin receptor gene (LEPR) overlapping with CNV_1815 showed remarkably higher copy number in Qinchuan than Nanyang (log2 (ratio) = -2.34988; P value = 1.53E-102). Further qPCR and association analysis investigated that the copy number of the LEPR gene presented positive correlations with transcriptional expression and phenotypic traits, suggesting the LEPR CNV may contribute to the higher fat deposition in muscles of Qinchuan cattle. Our findings provide evidence that the distinct phenotypes of Nanyang and Qinchuan breeds may be due to the different genetic variations including SNPs, indels and CNV.