Keren Long

Whole-genome sequencing of Berkshire (European native pig) provides insights into its origin and domestication

Domesticated organisms have experienced strong selective pressures directed at genes or genomic regions controlling traits of biological, agricultural or medical importance. The genome of native and domesticated pigs provide a unique opportunity for tracing the history of domestication and identifying signatures of artificial selection. Here we used whole-genome sequencing to explore the genetic relationships among the European native pig Berkshire and breeds that are distributed worldwide, and to identify genomic footprints left by selection during the domestication of Berkshire. Numerous nonsynonymous SNPs-containing genes fall into olfactory-related categories, which are part of a rapidly evolving superfamily in the mammalian genome. Phylogenetic analyses revealed a deep phylogenetic split between European and Asian pigs rather than between domestic and wild pigs. Admixture analysis exhibited higher portion of Chinese genetic material for the Berkshire pigs, which is consistent with the historical record regarding its origin. Selective sweep analyses revealed strong signatures of selection affecting genomic regions that harbor genes underlying economic traits such as disease resistance, pork yield, fertility, tameness and body length. These discoveries confirmed the history of origin of Berkshire pig by genome-wide analysis and illustrate how domestication has shaped the patterns of genetic variation.

Intrinsic Features in MicroRNA Transcriptomes Link Porcine Visceral Rather than Subcutaneous Adipose Tissues to Metabolic Risk

MicroRNAs (miRNAs) are non-coding small RNA ∼22 nucleotides in length that can regulate the expression of a wide range of coding genes at the post-transcriptional level. Visceral adipose tissues (VATs) and subcutaneous adipose tissues (SATs), the two main fat compartments in mammals, are anatomically, physiologically, metabolically, and clinically distinct. Various studies of adipose tissues have focused mainly on DNA methylation, and mRNA and protein expression, nonetheless little research sheds directly light on the miRNA transcriptome differences between these two distinct adipose tissue types. Here, we present a comprehensive investigation of miRNA transcriptomes across six variant porcine adipose tissues by small RNA-sequencing. We identified 219 known porcine miRNAs, 97 novel miRNA*s, and 124 miRNAs that are conserved to other mammals. A set of universally abundant miRNAs (i.e., miR-148a-3p, miR-143-3p, miR-27b-3p, miR-let-7a-1-5p, and miR-let-7f-5p) across the distinct adipose tissues was found. This set of miRNAs may play important housekeeping roles that are involved in adipogenesis. Clustering analysis indicated significant variations in miRNA expression between the VATs and SATs, and highlighted the role of the greater omentum in responding to potential metabolic risk because of the observed enrichment in this tissue of the immune- and inflammation-related miRNAs, such as the members of miR-17-92 cluster and miR-181 family. Differential expression of the miRNAs between the VATs and SATs, and miRNA target prediction analysis revealed that the VATs-specific enriched miRNAs were associated mainly with immune and inflammation responses. In summary, the differences of miRNA expression between the VATs and SATs revealed some of their intrinsic differences and indicated that the VATs might be closely associated with increased risk of metabolic disorders.

Exosomal microRNAs in giant panda (Ailuropoda melanoleuca) breast milk: potential maternal regulators for the development of newborn cubs

The physiological role of miRNAs is widely understood to include fine-tuning the post-transcriptional regulation of a wide array of biological processes. Extensive studies have indicated that exosomal miRNAs in the bodily fluids of various organisms can be transferred between living cells for the delivery of gene silencing signals. Here, we illustrated the expression characteristics of exosomal miRNAs in giant panda breast milk during distinct lactation periods and highlighted the enrichment of immune- and development-related endogenous miRNAs in colostral and mature giant panda milk. These miRNAs are stable, even under certain harsh conditions, via the protection of extracellular vesicles. These findings indicate that breast milk may facilitate the dietary intake of maternal miRNAs by infants for the regulation of postnatal development. We also detected exogenous plant miRNAs from the primary food source of the giant panda (bamboo) in the exosomes of giant panda breast milk that were associated with regulatory roles in basic metabolism and neuron development. This result suggested that dietary plant miRNAs are absorbed by host cells and subsequently secreted into bodily fluids as potential cross-kingdom regulators. In conclusion, exosomal miRNAs in giant panda breast milk may be crucial maternal regulators for the development of intrinsic ‘slink’ newborn cubs.

Detection of genetic diversity and selection at the coding region of the melanocortin receptor 1 (MC1R) gene in Tibetan pigs and Landrace pigs.

Domestication and subsequent selective pressures have produced a large variety of pig coat colors in different regions and breeds. The melanocortin 1 receptor (MC1R) gene plays a crucial role in determining coat color of mammals. Here, we investigated genetic diversity and selection at the coding region of the porcine melanocortin receptor 1 (MC1R) in Tibetan pigs and Landrace pigs. By contrast, genetic variability was much lower in Landrace pigs than in Tibetan pigs. Meanwhile, haplotype analysis showed that Tibetan pigs possessed shared haplotypes, suggesting a possibility of recent introgression event by way of crossbreeding with neighboring domestic pigs or shared ancestral polymorphism. Additionally, we detected positive selection at the MC1R in both Tibetan pigs and Landrace pigs through the dN/dS analysis. These findings suggested that novel phenotypic change (dark coat color) caused by novel mutations may help Tibetan pigs against intensive solar ultraviolet (UV) radiation and camouflage in wild environment, whereas white coat color in Landrace were intentionally selected by human after domestication. Furthermore, both the phylogenetic analysis and the network analysis provided clues that MC1R in Asian and European wild boars may have initially experienced different selective pressures, and MC1R alleles diversified in modern domesticated pigs.

Overexpression of Exosomal Cardioprotective miRNAs Mitigates Hypoxia-Induced H9c2 Cells Apoptosis

Recent evidence suggests that hypoxia caused by acute myocardial infarction can induce cardiomyocyte apoptosis. Exosomes are signalling mediators that contribute to intercellular communication by transporting cytosolic components including miRNAs, mRNAs, and proteins. However, the systemic regulation and function of exosomal miRNAs in hypoxic cardiomyocytes are currently not well understood. Here, we used small RNA sequencing to investigate the effects of hypoxia stress on miRNAome of rat cardiomyoblast cells (H9c2) and corresponding exosomes. We identified 92 and 62 miRNAs in cells and exosomes, respectively, that were differentially expressed between hypoxia and normoxia. Hypoxia strongly modulated expression of hypoxia-associated miRNAs in H9c2 cells, and altered the miRNAome of H9c2 cells-derived exosomes. Functional enrichment analysis revealed extensive roles of differentially expressed exosomal miRNAs in the HIF-1 signalling pathway and in apoptosis-related pathways including the TNF, MAPK, and mTOR pathways. Furthermore, gain- and loss-of-function analysis demonstrated potential anti-apoptotic effects of the hypoxia-induced exosomal miRNAs, including miR-21-5p, miR-378-3p, miR-152-3p, and let-7i-5p; luciferase reporter assay confirmed that Atg12 and Faslg are targets of miR-152-3p and let-7i-5p, respectively. To summarize, this study revealed that hypoxia-induced exosomes derived from H9c2 cells loaded cardioprotective miRNAs, which mitigate hypoxia-induced H9c2 cells apoptosis.

Dynamic gene expression profiles during postnatal development of porcine subcutaneous adipose

A better understanding of the control of lipogenesis is of critical importance for both human and animal physiology. This requires a better knowledge of the changes of gene expression during the process of adipose tissue development. Thus, the objective of the current study was to determine the effects of development on subcutaneous adipose tissue gene expression in growing and adult pigs. Here, we present a comprehensive investigation of mRNA transcriptomes in porcine subcutaneous adipose tissue across four developmental stages using digital gene expression profiling. We identified 3,274 differential expressed genes associated with oxidative stress, immune processes, apoptosis, energy metabolism, insulin stimulus, cell cycle, angiogenesis and translation. A set of universally abundant genes (ATP8, COX2, COX3, ND1, ND2, SCD and TUBA1B) was found across all four developmental stages. This set of genes may play important roles in lipogenesis and development. We also identified development-related gene expression patterns that are linked to the different adipose phenotypes. We showed that genes enriched in significantly up-regulated profiles were associated with phosphorylation and angiogenesis. In contrast, genes enriched in significantly down-regulated profiles were related to cell cycle and cytoskeleton organization, suggesting an important role for these biological processes in adipose growth and development. These results provide a resource for studying adipose development and promote the pig as a model organism for researching the development of human obesity, as well as being used in the pig industry.

Expression profiles of microRNAs in oxidized low-density lipoprotein-stimulated RAW 264.7 cells

Macrophage-derived foam cells were one of the hallmarks of atherosclerosis, and microRNAs played an important role in the formation of foam cells. In order to explore the roles of miRNA in the formation of foam cells, we investigated miRNA expression profiles in foam cells through high-throughput sequencing technology. A total of 84 miRNAs were differentially expressed between RAW 264.7 macrophages and foam cells induced by ox-LDL. Thirty miRNAs were upregulated and 54 miRNAs were downregulated. GO terms and KEGG pathways analysis revealed that the target genes of most of DE miRNAs were mainly enriched in “cell differentiation,” “endocytosis,” “MAPK signaling pathway,” and “FoxO signaling pathway.” The target genes of some DE miRNAs were enriched in “Insulin signaling pathway,” “Hippo signaling pathway,” “TNF signaling pathway,” “NF-kappa B signaling pathway,” and “cell death.” Using bioinformatics analyses and dual-luciferase reporter assays, we found that miR-28a-5p and miR-30c-1-3p directly inhibited LRAD3 and LOX-1 mRNA expression through targeting the 3’UTR of LRAD3 and LOX-1 mRNA, respectively. Our study indicates that miRNAs are extensively involved in the formation of foam cells, and provides a valuable resource for further study the role of miRNAs in atherosclerosis.

Comparative analysis of the microRNA transcriptome between yak and cattle provides insight into high-altitude adaptation

Extensive and in-depth investigations of high-altitude adaptation have been carried out at the level of morphology, anatomy, physiology and genomics, but few investigations focused on the roles of microRNA (miRNA) in high-altitude adaptation. We examined the differences in the miRNA transcriptomes of two representative hypoxia-sensitive tissues (heart and lung) between yak and cattle, two closely related species that live in high and low altitudes, respectively. In this study, we identified a total of 808 mature miRNAs, which corresponded to 715 pre-miRNAs in the two species. The further analysis revealed that both tissues showed relatively high correlation coefficient between yak and cattle, but a greater differentiation was present in lung than heart between the two species. In addition, miRNAs with significantly differentiated patterns of expression in two tissues exhibited co-operation effect in high altitude adaptation based on miRNA family and cluster. Functional analysis revealed that differentially expressed miRNAs were enriched in hypoxia-related pathways, such as the HIF-1α signaling pathway, the insulin signaling pathway, the PI3K-Akt signaling pathway, nucleotide excision repair, cell cycle, apoptosis and fatty acid metabolism, which indicated the important roles of miRNAs in high altitude adaptation. These results suggested the diverse degrees of miRNA transcriptome variation in different tissues between yak and cattle, and suggested extensive roles of miRNAs in high altitude adaptation.

Identification of exosome-like nanoparticle-derived microRNAs from 11 edible fruits and vegetables

Edible plant-derived exosome-like nanoparticles (EPDELNs) are novel naturally occurring plant ultrastructures that are structurally similar to exosomes. Many EPDELNs have anti-inflammatory properties. MicroRNAs (miRNAs) play a critical role in mediating physiological and pathological processes in animals and plants. Although miRNAs can be selectively encapsulated in extracellular vesicles, little is known about their expression and function in EPDELNs. In this study, we isolated nanovesicles from 11 edible fruits and vegetables and subjected the corresponding EPDELN small RNA libraries to Illumina sequencing. We identified a total of 418 miRNAs—32 to 127 per species—from the 11 EPDELN samples. Target prediction and functional analyses revealed that highly expressed miRNAs were closely associated with the inflammatory response and cancer-related pathways. The 418 miRNAs could be divided into three classes according to their EPDELN distributions: 26 “frequent” miRNAs (FMs), 39 “moderately present” miRNAs (MPMs), and 353 “rare” miRNAs (RMs). FMs were represented by fewer miRNA species than RMs but had a significantly higher cumulative expression level. Taken together, our in vitro results indicate that miRNAs in EPDELNs have the potential to regulate human mRNA.

Analysis of mitochondrial DNA sequence and copy number variation across five high-altitude species and their low-altitude relatives

Abstract High-altitude inhospitable environments impose a formidable life challenge for the local animals. Training and exposure to high-altitude environments produce both distinct physiological and phenotypic characteristics. The mitochondrion, an organelle crucial for the energy production, plays an important role in hypoxia adaptation. In this study, we investigated the mitochondrial DNA (mtDNA) polymorphism and copy number variation between the population pairs from distinct altitudes across the multi-species. Higher mitochondrial DNA control region’s genetic diversity is conspicuous in high-altitude animals versus low-altitude relatives. We also found an accordant decrease of mtDNA copy number in most of the tissues from high-altitude animals. Compared to mammals, chickens have significantly distinct mitogenomic characteristics, and more significant changes in the skeletal muscle mtDNA copy number between high- and low-altitude individuals. Our study catches a snapshot of the biological similarities and differences in the mitochondrial high-altitude acclimation across the species.