Comprehensive Gene and microRNA Expression Profiling Reveals a Role for microRNAs in Human Liver Development

Background Understanding the expression of non-coding RNA in the liver during embryonic development provides important insights into liver diseases. Therefore, we investigated circular RNA (circRNA) roles in human liver development, an unexplored research domain. Methods Using high-throughput sequencing and bioinformatics, we analysed foetal liver samples across developmental stages (7–20 weeks post-conception). Differentially expressed (DE) genes were identified and subjected to enrichment analysis using Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG), and Disease Ontology (DO). Modular analysis was performed using the Search Tool for Retrieval of Interacting Genes (STRING), followed by construction of a protein-protein interaction (PPI) network using Cytoscape software. The key genes were screened using Molecular Complex Detection (MCODE). The mRNA levels of hub genes were validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results There were 645 DE circRNAs and 5,145 DE mRNAs between human livers at the three growth stages (HB, EH, and LH). It was found that the activity of circRNAs was boosted remarkably in the hepatoblastic stage. Enrichment analysis found they mainly involved in nervous system regulation of liver function, embryonic organ development and digestive system development. In addition, DE circRNAs were primarily involved in the PI3K-AKT, MAPK and calcium pathways, potentially contributing to adult liver diseases. Notably, only hsa_circ_001471 and novel_circ_017382 were simultaneously identified at all stages and were persistently downregulated. A co-expression regulatory network involving these circRNAs was established. Three hub genes (LGR5, FOXL1 and RSPO3) were identified from the PPI network of 167 genes and may play key roles in human liver development. The RT-qPCR validation results were in agreement with the sequencing data. Conclusions Our findings provide the first insights into the roles and regulatory networks of circRNAs in human liver development, laying the groundwork for further investigations of molecular and signalling networks.

Human liver development commences during the third to fourth week of gestation. Endodermal cells give rise to hepatic cords representing liver progenitor cells. These progenitor cells are pluripotent because they differentiate along both the hepatocyte lineage (hepatoblasts) and the biliary epithelial cell lineage (ductal plate cells). In addition, ductal plate cells give rise to peri-biliary glands and in some cases to ectopic exocrine pancreatic tissue. Thus, the progenitor cells in the human fetal liver appear to have a broad differentiation potential similar to the putative hepatic stem cells or oval cells in the rat liver, which may develop into hepatocytes, biliary epithelial cells, pancreatic cells, and intestinal cells. Over the last decade, the existence and location of hepatic stem cells has been hotly debated and therefore hepatic development and the pluripotential capacities of liver progenitor cells have generated much interest. This chapter concisely presents the current stage of understanding of human liver development.

The epidermal growth factor receptor (EGFR) is frequently overexpressed in cancer and is an important therapeutic target. Aberrant expression and function of microRNAs have been associated with tumorigenesis. Bioinformatic predictions suggest that the human EGFR mRNA 3'-untranslated region contains three microRNA-7 (miR-7) target sites, which are not conserved across mammals. We found that miR-7 down-regulates EGFR mRNA and protein expression in cancer cell lines (lung, breast, and glioblastoma) via two of the three sites, inducing cell cycle arrest and cell death. Because miR-7 was shown to decrease EGFR mRNA expression, we used microarray analysis to identify additional mRNA targets of miR-7. These included Raf1 and multiple other genes involved in EGFR signaling and tumorigenesis. Furthermore, miR-7 attenuated activation of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2, two critical effectors of EGFR signaling, in different cancer cell lines. These data establish an important role for miR-7 in controlling mRNA expression and indicate that miR-7 has the ability to coordinately regulate EGFR signaling in multiple human cancer cell types.

Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based invitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations.

One potential role of miRNAs is to buffer variation in gene expression, although conflicting results have been reported. To investigate the buffering role of miRNAs in response to Salmonella infection in pigs, we sequenced miRNA and mRNA in whole blood from 15 pig samples before and after Salmonella challenge. By analyzing inter-individual variation in gene expression patterns, we found that for moderately and lowly expressed genes, putative miRNA targets showed significantly lower expression variance compared with non-miRNA-targets. Expression variance between highly expressed miRNA targets and non-miRNA-targets was not significantly different. Further, miRNA targets demonstrated significantly reduced variance after challenge whereas non-miRNA-targets did not. RNA binding proteins (RBPs) are significantly enriched among the miRNA targets with dramatically reduced variance of expression after Salmonella challenge. Moreover, we found evidence that targets of young (less-conserved) miRNAs showed lower expression variance compared with targets of old (evolutionarily conserved) miRNAs. These findings point to the importance of a buffering effect of miRNAs for relatively lowly expressed genes, and suggest that the reduced expression variation of RBPs may play an important role in response to Salmonella infection.

Next generation sequencing of the Ago2 interacting transcriptome identified chemokine family members as novel targets of neuronal microRNAs in hepatic stellate cells

Retained introns increase putative microRNA targets within 3′ UTRs of human mRNA

Cell-autonomous and signal-dependent expression of liver and intestine marker genes in pluripotent precursor cells from Xenopus embryos

MicroRNAs (miRNAs) are short endogenous noncoding RNAs that bind to target mRNAs, usually resulting in degradation and translational repression. Identification of miRNA targets is crucial for deciphering functional roles of the numerous miRNAs that are rapidly generated by sequencing efforts. Computational prediction methods are widely used for high-throughput generation of putative miRNA targets. We review a comprehensive collection of 38 miRNA sequence-based computational target predictors in animals that were developed over the past decade. Our in-depth analysis considers all significant perspectives including the underlying predictive methodologies with focus on how they draw from the mechanistic basis of the miRNA-mRNA interaction. We also discuss ease of use, availability, impact of the considered predictors and the evaluation protocols that were used to assess them. We are the first to comparatively and comprehensively evaluate seven representative methods when predicting miRNA targets at the duplex and gene levels. The gene-level evaluation is based on three benchmark data sets that rely on different ways to annotate targets including biochemical assays, microarrays and pSILAC. We offer practical advice on selection of appropriate predictors according to certain properties of miRNA sequences, characteristics of a specific application and desired levels of predictive quality. We also discuss future work related to the design of new models, data quality, improved usability, need for standardized evaluation and ability to predict mRNA expression changes.

In this issue of Hepatology, Devanand Sarkar and coworkers report that AEG-1, a gene located at chromosomal region 8q22, cooperates with c-MYC (8q24) to induce a highly metastatic form of hepatocellular carcinoma (HCC) in transgenic mice.[1] The authors used the ALB (albumin) promoter to drive selective expression of these two 8q genes in mature hepatocytes, under conditions where MYC expression alone is sufficient to induce HCC, albeit a less-aggressive form without distant metastasis. Interestingly, expression of AEG-1 and MYC forms a positive feedback loop, in that each promotes the expression of the other, presumably playing a normal interacting role in liver development that is hijacked during hepatocarcinogenesis. Their oncogenic cooperation has an additional interesting feature in that the combination of AEG-1 and MYC, but neither gene alone, induces expression of maternally imprinted long noncoding RNAs (lncRNAs) RIAN and MEG3. The authors go on to show that expression of these lncRNAs play a functional role in AEG-1/MYC-induced malignancy.[1] As with the study of microRNAs a few years back, the study of lncRNAs is becoming increasingly central to current research in hepatocarcinogenesis.

In nonalcoholic fatty liver disease, hepatic gene expression and fatty acid (FA) composition have been reported independently, but a comprehensive gene expression profiling in relation to FA composition is lacking. The aim was to assess this relationship. In a cross-sectional study, hepatic gene expression (Illumina Microarray) was first compared among 20 patients with simple steatosis (SS), 19 with nonalcoholic steatohepatitis (NASH), and 24 healthy controls. The FA composition in hepatic total lipids was compared between SS and NASH, and associations between gene expression and FAs were examined. Gene expression differed mainly between healthy controls and patients (SS and NASH), including genes related to unsaturated FA metabolism. Twenty-two genes were differentially expressed between NASH and SS; most of them correlated with disease severity and related more to cancer progression than to lipid metabolism. Biologically active long-chain polyunsaturated FAs (PUFAs; eicosapentaenoic acid + docosahexaenoic acid, arachidonic acid) in hepatic total lipids were lower in NASH than in SS. This may be related to overexpression of FADS1, FADS2, and PNPLA3. The degree and direction of correlations between PUFAs and gene expression were different among SS and NASH, which may suggest that low PUFA content in NASH modulates gene expression in a different way compared with SS or, alternatively, that gene expression influences PUFA content differently depending on disease severity (SS versus NASH). Well-defined subjects with either healthy liver, SS, or NASH showed distinct hepatic gene expression profiles including genes involved in unsaturated FA metabolism. In patients with NASH, hepatic PUFAs were lower and associations with gene expression were different compared to SS.

PurposeTo investigate the role of miR-29b on the changes in expression of genes involved in the synthesis and deposition of extracellular matrix (ECM) induced by chronic oxidative stress in human trabecular meshwork cells (HTM).MethodsChanges in gene expression induced by miR-29b in HTM cells were evaluated by gene array analysis using Affymetrix U133A2 arrays and confirmed by quantitative–PCR. Pathway analysis was conducted using Metacore. Targeting of miR-29b to the 3’-untranslated region of three novel putative targets was evaluated using the Psicheck luciferase system. Chronic oxidative stress was induced by incubation at 40% oxygen for 4–5 days, using cultures incubated at 5% oxygen as controls. Changes in expression in microRNA or gene expression were analyzed by Q-PCR. Cell viability was measured by lactate dehydrogenase release.ResultsTransfection of HTM cells with miR-29b mimic resulted in downregulation of multiple ECM components, including collagens (COL1A1, COL1A2, COL4A1, COL5A1, COL5A2, COL3A1) LAMC1, and FBN as well as several genes involved in ECM deposition and remodeling, such as SPARC/osteonectin. Three additional genes, BMP1, ADAM12, and NKIRAS2, were identified as direct targets of miR-29b. Chronic oxidative stress induced a significant downregulation of miR-29b in two HTM cell lines that was associated with increased expression of several ECM genes known to be regulated by miR-29b. The increase in expression of these genes was inhibited by transfection with miR-29b mimic. MiR-29b increased cell viability under both chronic oxidative stress and physiologic oxygen concentrations.ConclusionsMiR-29b negatively regulates the expression of multiple genes involved in the synthesis and deposition of ECM in trabecular meshwork (TM) cells. Downregulation of miR-29b might contribute to increased expression of several ECM genes under chronic oxidative stress conditions. The balance between the activation of ECM production induced by oxidative stress and the protective effects of miR-29b could be a relevant factor in understanding how oxidative damage may lead to increased deposition of ECM in the TM and contribute to the elevation of intra-ocular pressure in glaucoma.

BackgroundMicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia.ResultsWe identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish.ConclusionsThe sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2636-z) contains supplementary material, which is available to authorized users.

Tumor-associated macrophages (TAMs) infiltrate both mouse and human tumors, typically in high numbers, and display complex interactions with cancer cells, endothelial cells (ECs) and other immune cells. TAMs comprise molecularly distinct subsets. Two such subsets, reminiscent of Th1 and Th2-activated macrophages, are frequently denoted M1 and M2-like TAMs, respectively. Whereas M2-like TAMs are immunosuppressive, proangiogenic and largely tumor-promoting; M1-like TAMs display a more proinflammatory profile. microRNAs (miRNAs), a large class of small noncoding RNAs that modulate gene expression post-transcriptionally, are expressed at high levels in macrophages and are involved in macrophage differentiation and activation. However, beyond specific miRNAs, the significance of general miRNA activity for TAM's immunological and tumor-regulatory functions remains unclear. Hence, the aim of this thesis is to characterize the role of miRNAs in TAMs, both endogenously and as secreted molecules. In the first part of this thesis, I used a genetic mouse model to conditionally delete the miRNA-processing enzyme Dicer in TAMs, and found that it greatly amplified their M1-like programming and inhibited tumor growth. TAM skewing fostered the recruitment and activation of cytotoxic T cells (CTLs), which exacerbated M1-like TAM polarization through interferon-gamma. Remarkably, TAM's DICER status determined pro versus anti-tumoral outcome of TAM or CTL depletion, and controlled response to immunotherapy. Finally, I found that the genetic rescue of the miRNA Let-7 in Dicer-deficient TAMs was sufficient to restore the M2-like TAM phenotype and decrease CTLs. These findings indicate that DICER/Let-7 activity opposes immunostimulatory TAM activation, with potential therapeutic implications. In the second part, I investigated the role of secreted, macrophage-derived miRNAs and their transfer to other cells via exosomes. Exosomes are small vesicles that have emerged as mediators of intercellular communication. They are released in the extracellular space where they can fuse with, and deliver their cargo to, other cells. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms by which miRNAs are sorted to exosomes and the significance of miRNA transfer to other cells are unclear. RNA profiling of macrophages and their exosomes showed that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetic perturbation of the expression of individual miRNAs, or their targeted transcripts, promoted bidirectional miRNA relocation from the cell cytoplasm (site of miRNA activity) to multivesicular-bodies (sites of exosome biogenesis) and controlled miRNA sorting to exosomes. The use of Dicer-deficient cells and reporter lentiviral vectors for miRNA activity further showed that exosomal miRNAs are transferred from macrophages to ECs to detectably repress target sequences. These findings indicate that macrophages can communicate with ECs via transfer of exosomes and may mediate some of their proangiogenic interactions by doing so. This work provides new insights into the role of miRNAs in TAMs as well as their complex interactions with the tumor microenvironment. It also highlights the importance of miRNAs in TAMs, which may represent novel therapeutic targets to reprogram TAMs.

Aims/hypothesisType 2 diabetes is characterised by progressive beta cell dysfunction, with changes in gene expression playing a crucial role in its development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and therefore alterations in miRNA levels may be involved in the deterioration of beta cell function.MethodsGlobal TaqMan arrays and individual TaqMan assays were used to measure islet miRNA expression in discovery (n = 20) and replication (n = 20) cohorts from individuals with and without type 2 diabetes. The role of specific dysregulated miRNAs in regulating insulin secretion, content and apoptosis was subsequently investigated in primary rat islets and INS-1 cells. Identification of miRNA targets was assessed using luciferase assays and by measuring mRNA levels.ResultsIn the discovery and replication cohorts miR-187 expression was found to be significantly increased in islets from individuals with type 2 diabetes compared with matched controls. An inverse correlation between miR-187 levels and glucose-stimulated insulin secretion (GSIS) was observed in islets from normoglycaemic donors. This correlation paralleled findings in primary rat islets and INS-1 cells where overexpression of miR-187 markedly decreased GSIS without affecting insulin content or apoptotic index. Finally, the gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known regulator of insulin secretion, was identified as a direct target of miR-187 and displayed reduced expression in islets from individuals with type 2 diabetes.Conclusions/interpretationOur findings suggest a role for miR-187 in the blunting of insulin secretion, potentially involving regulation of HIPK3, which occurs during the pathogenesis of type 2 diabetes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-3089-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users.