Characterization of the microRNA Expression Profiles in the Goat Kid Liver

The liver is the largest digestive gland in goats with an important role in early metabolic function development. MicroRNAs (miRNA) are crucial for regulating the development and metabolism in the goat liver. In the study, we sequenced the miRNAs in the liver tissues of the goat kid to further research their regulation roles in early liver development. The liver tissues were procured at 5-time points from the Laiwu black goats of 1 day (D1), 2 weeks (W2), 4 weeks (W4), 8 weeks (W8), and 12 weeks (W12) after birth, respectively with five goats per time point, for a total of 25 goats. Our study identified 214 differential expression miRNAs, and the expression patterns of 15 randomly selected miRNAs were examined among all five age groups. The Gene ontology annotation results showed that differential expression miRNA (DE miRNA) target genes were significantly enriched in the fatty acid synthase activity, toxin metabolic process, cell surface, and antibiotic metabolic process. The KEGG analysis result was significantly enriched in steroid hormone synthesis and retinol metabolism pathways. Further miRNA-mRNA regulation network analysis reveals 9 differently expressed miRNA with important regulation roles. Overall, the DE miRNAs were mainly involved in liver development, lipid metabolism, toxin related metabolism-related biological process, and pathways. Our results provide new information about the molecular mechanisms and pathways in the goat kid liver development.

Genome-wide identification of microRNA targets reveals positive regulation of the Hippo pathway by miR-122 during liver development

Liver development is a highly complex process that is regulated by the orchestrated interplay of epigenetic regulators, transcription factors, and microRNAs (miRNAs). Owing to the lack of global in vivo targets of all miRNAs during liver development, the mechanisms underlying the dynamic control of hepatocyte differentiation by miRNAs remain elusive. Here, using Argonaute (Ago) high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse liver at different developmental stages, we characterized massive Ago-binding RNAs and obtained a genome-wide map of liver miRNA-mRNA interactions. The dynamic changes of five clusters of miRNAs and their potential targets were identified to be differentially involved at specific stages, a dozen of high abundant miRNAs and their epigenetic regulation by super-enhancer were found during liver development. Remarkably, miR-122, a liver-specific and most abundant miRNA in newborn and adult livers, was found by its targetome and pathway reporter analyses to regulate the Hippo pathway, which is crucial for liver size control and homeostasis. Mechanistically, we further demonstrated that miR-122 negatively regulates the outcomes of the Hippo pathway transcription factor TEAD by directly targeting a number of hippo pathway regulators, including the coactivator TAZ and a key factor of the phosphatase complex PPP1CC, which contributes to the dephosphorylation of YAP, another coactivator downstream of the Hippo pathway. This study identifies for the first time the genome-wide miRNA targetomes during mouse liver development and demonstrates a novel mechanism of terminal differentiation of hepatocytes regulated by the miR-122/Hippo pathway in a coordinated manner. As the Hippo pathway plays important roles in cell proliferation and liver pathological processes like inflammation, fibrosis, and hepatocellular carcinoma (HCC), our study could also provide a new insight into the function of miR-122 in liver pathology.

Functional Recellularization of Acellular Rat Liver Scaffold by Induced Pluripotent Stem Cells: Molecular Evidence for Wnt/B-Catenin Upregulation

Background. Liver transplantation remains the only viable therapy for liver failure but has a severely restricted utility. Here, we aimed to decellularize rat livers to form acellular 3D bio-scaffolds suitable for seeding with induced pluripotent cells (iPSCs) as a tool to investigate the role of Wnt/β-catenin signaling in liver development and generation. Methods. Dissected rat livers were randomly divided into three groups: I (control); II (decellularized scaffolds) and III (recellularized scaffolds). Liver decellularization was established via an adapted perfusion procedure and assessed through the measurement of extracellular matrix (ECM) proteins and DNA content. Liver recellularization was assessed through histological examination and measurement of transcript levels of Wnt/β-catenin pathway, hepatogenesis, liver-specific microRNAs and growth factors essential for liver development. Adult rat liver decellularization was confirmed by the maintenance of ECM proteins and persistence of growth factors essential for liver regeneration. Results. iPSCs seeded rat decellularized livers displayed upregulated transcript expression of Wnt/β-catenin pathway-related, growth factors, and liver specification genes. Further, recellularized livers displayed restored liver-specific functions including albumin secretion and urea synthesis. Conclusion. This establishes proof-of-principle for the generation of three-dimensional liver organ scaffolds as grafts and functional re-establishment.

Connexin Expression Is Altered in Liver Development of Yotari (dab1 -/-) Mice

Disabled-1 (Dab1) protein is an intracellular adaptor of reelin signaling required for prenatal neuronal migration, as well as postnatal neurotransmission, memory formation and synaptic plasticity. Yotari, an autosomal recessive mutant of the mouse Dab1 gene is recognizable by its premature death, unstable gait and tremor. Previous findings are mostly based on neuronal abnormalities caused by Dab1 deficiency, but the role of the reelin signaling pathway in nonneuronal tissues and organs has not been studied until recently. Hepatocytes, the most abundant cells in the liver, communicate via gap junctions (GJ) are composed of connexins. Cell communication disruption in yotari mice was examined by analyzing the expression of connexins (Cxs): Cx26, Cx32, Cx37, Cx40, Cx43 and Cx45 during liver development at 13.5 and 15.5 gestation days (E13.5 and E15.5). Analyses were performed using immunohistochemistry and fluorescent microscopy, followed by quantification of area percentage covered by positive signal. Data are expressed as a mean±SD and analyzed by one-way ANOVA. All Cxs examined displayed a significant decrease in yotari compared to wild type (wt) individuals at E13.5. Looking at E15.5 we have similar results with exception of Cx37 showing negligible expression in wt. Channels formation triggered by pathological stimuli, as well as propensity to apoptosis, was studied by measuring the expression of Pannexin1 (Panx1) and Apoptosis-inducing factor (AIF) through developmental stages mentioned above. An increase in Panx1 expression of E15.5 yotari mice, as well as a strong jump of AIF in both phases suggesting that yotari mice are more prone to apoptosis. Our results emphasize the importance of gap junction intercellular communication (GJIC) during liver development and their possible involvement in liver pathology and diagnostics where they can serve as potential biomarkers and drug targets.

Integrated Analysis of Liver Transcriptome, miRNA, and Proteome of Chinese Indigenous Breed Ningxiang Pig in Three Developmental Stages Uncovers Significant miRNA–mRNA–Protein Networks in Lipid Metabolism

Liver is an important metabolic organ of mammals. During each transitional period of life, liver metabolism is programmed by a complex molecular regulatory system for multiple physiological functions, many pathways of which are regulated by hormones and cytokines, nuclear receptors, and transcription factors. To gain a comprehensive and unbiased molecular understanding of liver growth and development in Ningxiang pigs, we analyzed the mRNA, microRNA (miRNA), and proteomes of the livers of Ningxiang pigs during lactation, nursery, and fattening periods. A total of 22,411 genes (19,653 known mRNAs and 2758 novel mRNAs), 1122 miRNAs (384 known miRNAs and 738 novel miRNAs), and 1123 unique proteins with medium and high abundance were identified by high-throughput sequencing and mass spectrometry. We show that the differences in transcriptional, post-transcriptional, or protein levels were readily identified by comparing different time periods, providing evidence that functional changes that may occur during liver development are widespread. In addition, we found many overlapping differentially expressed genes (DEGs)/differentially expressed miRNAs (DEMs)/differentially expressed proteins (DEPs) related to glycolipid metabolism in any group comparison. These overlapping DEGs/DEMs/DGPs may play an important role in functional transformation during liver development. Short Time-series Expression Miner (STEM) analysis revealed multiple expression patterns of mRNA, miRNA, and protein in the liver. Furthermore, several diverse key Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including immune defense, glycolipid metabolism, protein transport and uptake, and cell proliferation and development, were identified by combined analysis of DEGs and DGPs. A number of predicted miRNA–mRNA–protein pairs were found and validated by qRT-PCR and parallel reaction monitoring (PRM) assays. The results provide new and important information about the genetic breeding of Ningxiang pigs, which represents a foundation for further understanding the molecular regulatory mechanisms of dynamic development of liver tissue, functional transformation, and lipid metabolism.

Functions of SMC2 in the Development of Zebrafish Liver

SMC2 (structural maintenance of chromosomes 2) is the core subunit of condensins, which play a central role in chromosome organization and segregation. However, the functions of SMC2 in embryonic development remain poorly understood, due to the embryonic lethality of homozygous SMC2−/− mice. Herein, we explored the roles of SMC2 in the liver development of zebrafish. The depletion of SMC2, with the CRISPR/Cas9-dependent gene knockout approach, led to a small liver phenotype. The specification of hepatoblasts was unaffected. Mechanistically, extensive apoptosis occurred in the liver of SMC2 mutants, which was mainly associated with the activation of the p53-dependent apoptotic pathway. Moreover, an aberrant activation of a series of apoptotic pathways in SMC2 mutants was involved in the defective chromosome segregation and subsequent DNA damage. Therefore, our findings demonstrate that SMC2 is necessary for zebrafish liver development.