Boxing Sun

A comparative profile of the microRNA transcriptome in immature and mature porcine testes using Solexa deep sequencing.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that play key roles in many diverse biological processes such as spermatogenesis. However, no study has been performed on the miRNA transcriptome of developing porcine testes. Here, we employed Solexa deep sequencing technology to extend the repertoire of porcine testis miRNAs and extensively compare the expression patterns of sexually immature and mature porcine testes. Solexa sequencing of two small RNA libraries derived from immature (30 days) and mature (180 days) pig testis samples yielded over 25 million high‐quality reads. Overall, the two developmental stages had significantly different small RNA compositions. A custom data analysis pipeline identified 398 known and/or homologous conserved porcine miRNAs, 15 novel pig‐specific miRNAs and 56 novel candidate miRNAs. We further observed multiple mature miRNA variants and identified a new bidirectional transcribed miRNA locus, ssc‐mir‐181a. A total of 122 miRNAs were differentially expressed in the immature and mature testes, and 10 were validated using quantitative RT‐PCR. Furthermore, GO and KEGG pathway analyses of the predicted miRNA targets further illustrate the likely roles for these differentially expressed miRNAs in spermatogenesis. This study is the first comparative profile of the miRNA transcriptome in immature and mature porcine testes using a deep sequencing approach, and it provides a useful resource for future studies on the role of miRNAs in spermatogenesis and male infertility treatment.

Analysis of DNA Methylation in Various Swine Tissues

DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total, 96,371 fragments, each representing a recognition site cleaved by either or both EcoRI + HpaII and EcoRI + MspI, the HpaII and MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle, 51.24% in the heart, 50.18% in the liver, 53.31% in the spleen, 51.97% in the lung, 51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277, 24,801 and 25,293, respectively. In addition, different methylation patterns were observed in seven tissues from pig, and almost all of the methylation patterns detected by F-MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrated that the F-MSAP technique can be adapted for use in large-scale DNA methylation detection in the pig genome.

Association of A-FABP gene polymorphism in intron 1 with meat quality traits in Junmu No. 1 white swine.

This study was designed to investigate the single nucleotide polymorphism (SNP) in intron 1 of the gene A-FABP in 127 Junmu No. 1 white swine using PCR-SSCP. The association between the polymorphism and meat quality traits was also studied. The cloning and sequencing results indicated that the polymorphism of intron 1 was due to a point mutation in position 3481bp of A-FABP, giving 3 genotypes (CC, CD and DD). Association analysis indicated that the polymorphism had a significant effect on marbling (P<0.05). Genotype DD had higher marbling than CD and CC, but the difference between CD and CC was no significant. Polymorphism had a highly significant effect on intramuscular fat (IMF) content (P<0.01). DD was higher than CD, which was higher than CC. No significant conclusions can be drawn regarding other traits. Immunoblot analysis of A-FABP levels was carried out on 3 different genotype individuals. Expression was markedly reduced in DD compared with genotype CC. Thus A-FABP may be a candidate gene or a quantitative trait locus-linked gene associated with meat quality traits.

MicroRNAs in the immune organs of chickens and ducks indicate divergence of immunity against H5N1 avian influenza

Chickens are susceptible to the highly pathogenic H5N1 strain of avian influenza virus (HPAIV), whereas ducks are not. Here, we used high‐throughput sequencing to analyse the microRNA expression in the spleen, thymus and bursa of Fabricius of H5N1‐HPAIV‐infected and non‐infected chickens and ducks. We annotated the genomic positions of duck microRNAs and we compared the microRNA repertoires of chickens and ducks. Our results showed that the microRNA expression patterns in the homologous immune organs of specific‐pathogen‐free (SPF) chickens and ducks diverge substantially. Moreover, there was larger divergence between the microRNA expression patterns in immune organs of HPAIV‐infected chickens than HPAIV‐infected ducks. Together, our results might help to elucidate the roles of microRNAs in the divergent immunity of chickens and ducks against H5N1 HPAIV.

A base substitution in the donor site of intron 12 of KIT gene is responsible for the dominant white coat colour of blue fox (Alopex lagopus).

The dominant white coat colour of farmed blue fox is inherited as a monogenic autosomal dominant trait and is suggested to be embryonic lethal in the homozygous state. In this study, the transcripts of KIT were identified by RT-PCR for a dominant white fox and a normal blue fox. Sequence analysis showed that the KIT transcript in normal blue fox contained the full-length coding sequence of 2919 bp (GenBank Acc. No KF530833), but in the dominant white individual, a truncated isoform lacking the entire exon 12 specifically co-expressed with the normal transcript. Genomic DNA sequencing revealed that a single nucleotide polymorphism (c.1867+1G>T) in intron 12 appeared only in the dominant white individuals and a 1-bp ins/del polymorphism in the same intron showed in individuals representing two different coat colours. Genotyping results of the SNP with PCR-RFLP in 185 individuals showed all 90 normal blue foxes were homozygous for the G allele, and all dominant white individuals were heterozygous. Due to the truncated protein with a deletion of 35 amino acids and an amino acid replacement (p.Pro623Ala) located in the conserved ATP binding domain, we propose that the mutant receptor had absent tyrosine kinase activity. These findings reveal that the base substitution at the first nucleotide of intron 12 of KIT gene, resulting in skipping of exon 12, is a causative mutation responsible for the dominant white phenotype of blue fox.

The effect of leader peptide mutations on the biological function of bovine myostatin gene.

The growth of muscle fibers can be negatively regulated by bovine myostatin. The first two exons of myostatin gene code for the N-propeptide and its third exon codes for the C-polypeptide. Myostatin is secreted as a latent configuration formed by dimerization of two matured C peptides non-covalently linked with the N terminal pro-peptide. Pro-peptide has two distinct functions in guiding protein folding and regulating biological activity of myostatin. When the structure of the leader peptide is altered via mutations resulting in more tight binding with the mature peptide, myostatin function is inhibited, resulting in the changes of P21 and CDK2 expression levels which are related to the regulation of cell cycle. In the present study, the coding region of bMSTN (bovine myostatin) gene was amplified and mutated (A224C and G938A) through fusion PCR, and the mutated bMSTN gene (bMSTN-mut) was inserted in frame into the pEF1a-IRES-DsRed-Express2 vector and transfected into bovine fibroblast cells. The expression levels of bMSTN-mut, P21 and CDK2 (cyclin dependent kinase 2) were examined with qPCR and Western-blotting. Changes in cell cycle after transfection were also analyzed with flow cytometry. The results indicated that leader peptide mutation resulted in down-regulation of P21 expression levels and up-regulation of CDK2 expression levels. The flow cytometry results showed that the proportion of cells in the G0/G1-phase was lower and that of cells in the S-phase was higher in bMSTN-mut transfected group than that in the control group. The proliferation rate of bMSTN-mut transfected cells was also significantly higher than that of the control cells. In conclusion, the studies have shown that the pEF1a-IRES-DsRed-Express2-bMSTN-mut recombinant plasmid could effectively promote the proliferation of bovine fibroblast cells. The site-directed mutagenesis of bMSTN gene leader peptide and in vitro expression in bovine fibroblast cells could be helpful to further the studies of bMSTN in regulating bovine muscle cell growth and development.

MiR-152 Regulates Apoptosis and Triglyceride Production in MECs via Targeting ACAA2 and HSD17B12 Genes

Mammary epithelial cells (MECs) affect milk production capacity during lactation and are critical for the maintenance of tissue homeostasis. Our previous studies have revealed that the expression of miR-152 was increased significantly in MECs of cows with high milk production. In the present study, bioinformatics analysis identified ACAA2 and HSD17B12 as the potential targets of miR-152, which were further validated by dual-luciferase repoter assay. In addition, the expressions of miR-152 was shown to be negatively correlated with levels of mRNA and protein of ACAA2, HSD17B12 genes by qPCR and western bot analysis. Furthermore, transfection with miR-152 significantly up-regulated triglyceride production, promoted proliferation and inhibited apoptosis in MECs. Furthermore, overexpression of ACAA2 and HSD17B12 could inhibit triglyceride production, cells proliferation and induce apoptosis; but sh234-ACAA2-181/sh234-HSD17B12-474 could reverse the trend. These findings suggested that miR-152 could significantly influence triglyceride production and suppress apoptosis, possibly via the expression of target genes ACAA2 and HSD17B12.

miR-375 down-regulation of the rearranged L-myc fusion and hypoxia-induced gene domain protein 1A genes and effects on Sertoli cell proliferation

Objective This study aimed to screen and identify the target genes of miR-375 in pig Sertoli (ST) cells and to elucidate the effect of miR-375 on the proliferation of ST cells. Methods In this study, bioinformatics software was used to predict and verify miR-375 target genes. Quantitative polymerase chain reaction (PCR) was used to detect the relationship between miR-375 and its target genes in ST cells. Enzyme-linked immunosorbent assay (ELISA) of rearranged L-myc fusion (RLF) and hypoxia-induced gene domain protein 1A (HIGD1A) was performed on porcine ST cells, which were transfected with a miR-375 mimics and inhibitor to verify the results. Dual luciferase reporter gene assays were performed to assess the interactions among miR-375, RLF, and HIGD1A. The effect of miR-375 on the proliferation of ST cells was analyzed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS). Results Five possible target genes of miR-375, including RLF, HIGD1A, colorectal cancer associated 2, POU class 3 homeobox 1, and WW domain binding protein 1 like, were found. The results of quantitative PCR suggested that mRNA expression of RLF and HIGD1A had a negative correlation with miR-375, indicating that RLF and HIGD1A are likely the target genes of miR-375. The ELISA results revealed that RLF and HIGD1A were negatively correlated with the miR-375 protein level. The luminescence results for the miR-375 group co-transfected with wild-type RLF and HIGD1A vector were significantly lower than those of the miR-375 group co-transfected with the blank vector or mutant RLF and HIGD1A vectors. The present findings suggest that RLF and HIGD1A are target genes of miR-375 and that miR-375 inhibits ST cell proliferation according to MTS analysis. Conclusion It was speculated that miR-375 affects cell proliferation through its target genes, which play an important role in the development of testicular tissue.

Identification and characterization of the cDNA sequence encoding amelogenin in rabbit (Oryctolagus cuniculus).

Amelogenins, the most abundant proteins in tooth enamel extracellular matrix (ECM), are essential for tooth amelogenesis. The nucleotide sequence of amelogenin gene (AMEL) for rabbit, as an important member of mammals and good continuously growing incisor model, is important for comparative and evolutional study. Previous studies about rabbit amelogenin proteins got no consensus yet even as to their existence or size. In this study, with combined usage of in silico and molecular cloning technologies, we identified sequences of two transcripts of rabbit amelogenin, resulting from the alternative splicing of the 45-bp exon 4. The coding regions of the two transcripts are of 567- and 522-bp, encoding 188 and 173 amino acids including a 17-residue signal peptide, respectively. Sequence analysis revealed that rabbit amelogenin features in extremely high GC-content in nucleotide sequence and Alanine content in protein sequence. Detailed comparison of amino acid sequence with other mammals showed that the rabbit amelogenin protein is conserved in the sites and regions important for protein functions. Overall, our results uncovered the mysteries about rabbit amelogenin and revealed its sequence peculiarities.

Differential Expression of miR-34c and Its Predicted Target Genes in Testicular Tissue at Different Development Stages of Swine

To verified the target genes of miR-34c, bioinformatics software was used to predict the targets of miR-34c. Three possible target genes of miR-34c related to spermatogenesis and male reproductive development: zinc finger protein 148 (ZNF148), kruppel-like factor 4 (KLF4), and platelet-derived growth factor receptor alpha (PDGFRA) were predicted. Then, the expression of miR-34c and its target genes were detected in swine testicular tissue at different developmental stages by quantitative polymerase chain reaction. The results suggested that the expression of PDGFRA has the highest negative correlation with miR-34c. Then immunohistochemical staining was done to observe the morphology of swine testicular tissue at 2-days and 3, 4, 5-months of age, which indicated that PDGFRA was mainly expressed in the support cells near the basement membrane during the early development stages of testicular tissue, but that the expression of PDGFRA was gradually reduced in later stages. Therefore, western blot analyzed that the highest expression of PDGFRA was generated in 2-days old testicular tissues and the expression levels reduced at 3 and 4-months old, which correlated with the results of immunohistochemical staining. In conclusion, PDGFRA is a target gene of miR-34c.