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Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most frequent cause of cancer-related death. The IRX5 transcription factor plays a different role in multiple cancers and contributes to the development of many tumours. However, little is known about the molecular mechanisms of IRX5 in HCC. In this study, we found that IRX5 was abnormally upregulated in HCC tissues compared with adjacent normal tissues. IRX5 promoted HCC cell proliferation and upregulated the expression of cyclin D1 and knockdown of IRX5 suppressed tumorigenicity in vivo. Furthermore, knockdown of IRX5 increased p53 and Bax expression and decreased Bcl-2 expression. Thus, IRX5 suppressed apoptosis in HCC cells by inhibiting the p53 signalling pathway, indicating its role as a treatment target for HCC. SIGNIFICANCE OF THE STUDY: Our study demonstrated that IRX5 was abnormally upregulated in HCC tissues compared with adjacent normal tissues. IRX5 promoted HCC cell proliferation and upregulated the expression of cyclin D1, and knockdown of IRX5 suppressed tumorigenicity in vivo. Furthermore, knockdown of IRX5 increased p53 and Bax expression and decreased Bcl-2 expression. IRX5 suppressed apoptosis in HCC cells by inhibiting the p53 signalling pathway, indicating its role as a treatment target for HCC.

We report on 2 cousins, a girl and a boy, born to first-cousin Lebanese parents with Hamamy syndrome, exhibiting developmental delay, intellectual disability, severe telecanthus, abnormal ears, dentinogenesis imperfecta, and bone fragility. Whole-exome sequencing studies performed on the 2 affected individuals and one obligate carrier revealed the presence of a homozygous c.503G>A (p.Arg168His) missense mutation in IRX5 in both sibs, not reported in any other family. Review of the literature and differential diagnoses are discussed.

Aims: several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function. Methods and results: using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. Conclusion: altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases., National Research Agency; European Union (EU); Horizon 2020; Marie Curie Actions International Incoming Fellowship FP7-PEOPLE-2012-IIF; La Fédération Française de Cardiologie; Fondation LefoulonDelalande; Eiffel Scholarship Programme of Excellence (Campus France), Doctoral School of Science and Technology-Lebanese University and The Fondation Genavie

Background/Aims: The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene was first found to be activated in colorectal neoplasia. Now, it also has been found to be upregulated in many other solid tumors. Whether CRNDE affects tumorigenesis remains unknown. Methods: We conducted bioinformatics, real-time polymerase chain reaction (PCR), Western blot analysis, cell proliferation assay, colony formation assay, wound healing assay, cell migration and invasion assays, RNA immunoprecipitation, and reporter vector construction and luciferase assays. Results: CRNDE was upregulated in hepatocellular carcinoma (HCC). The overexpression of CRNDE promoted HCC cellular proliferation, migration, and invasion in intro and in vivo, and acted as an oncogene in HCC progression. Furthermore, CRNDE impaired miR-136-5P expression in a RISC manner, and a reciprocal repression feedback loop was possible between CRNDE and miR-136-5P. We found that the neighboring mRNA of CRNDE was IRX5, and IRX5 increased the tumorigenicity of HCC cells. IRX5 was a potential downstream target gene of miR-136-5P. MiR-136 regulated IRX5 by interacting with its 3’UTR. In addition, miR-136-5P was involved in the CRNDE-regulated expression of IRX5. Conclusion: CRNDE acted as a tumor oncogene by exhibiting oncogenic properties of human HCC and revealed a novel CRNDE-miR-136-5P-IRX5 regulatory network in HCC. CRNDE may be considered to be a potential target for HCC therapies based on its ability to upregulate IRX5, and it deserves further investigation.

In the 21st century, obesity has become a serious problem because of increasing obese patients and numerous metabolic complications. The primary reasons for this situation are environmental and genetic factors. In 2007, FTO (fat mass and obesity associated) was the first gene identified through a genome-wide association study (GWAS) associated with obesity in humans. Subsequently, a cluster of single nucleotide polymorphisms (SNPs) in the first intron of the FTO gene was discovered to be associated with BMI and body composition. Various studies have explored the mechanistic basis behind this association. Thus, emerging evidence showed that FTO plays a key role regulating adipose tissue development and functions in body size and composition. Recent prevalent research topic concentrated in the three neighboring genes of FTO: RPGRIP1L, IRX3 and IRX5, as having a functional link between obesity-associated common variants within FTO and the observed human phenotypes. The purpose of this review is to present a comprehensive picture of the impact of FTO on obesity susceptibility and to illuminate these new studies of FTO function in adipose tissue.

The Iroquois homeobox ( Irx5) gene is essential in embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 protein is involved in regulation of the cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies using primary human endothelial cells, VSMCs, and intact carotid arteries to determine relative expression of Irx5 in the peripheral vasculature demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization, and the presence of IRX5 was examined by immunohistochemistry after 2 wk. Results indicate markedly elevated IRX5 signal at 14 days compared with uninjured controls. Total RNA was isolated from injured and uninjured arteries, and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression at 3–14 days postinjury compared with controls. Irx5 genetic gain- and loss-of-function studies using thymidine and 5-bromo-2′-deoxyuridine incorporation assays resulted in modulation of DNA synthesis in primary rat aortic VSMCs. Quantitative RT-PCR results revealed modulation of cyclin-dependent kinase inhibitor 1B ( p27kip1), E2F transcription factor 1 ( E2f1), and proliferating cell nuclear antigen ( Pcna) expression in Irx5-transduced VSMCs compared with controls. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation compared with control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S-phase cell cycle checkpoint control and apoptosis.

Objective A causal obesity risk variant in the FTO locus was recently shown to inhibit adipocyte thermogenesis via increased adipose expression of the homeobox transcription factors IRX3 and IRX5. However, causal effects of IRX5 on fat storage remain to be shown in vivo, and discovery of downstream mediators may open new therapeutic avenues. Methods 17 WT and 13 Irx5 knockout (KO) mice were fed low-fat control (Ctr) or high-fat (HF) diet for 10 weeks. Body weight, energy intake and fat mass were measured. Irx5-dependent gene expression was explored by transcriptome analysis of epididymal white adipose tissue (eWAT), confirmatory obesity-dependent expression in human adipocytes in vivo, and in vitro knock-down, overexpression and transcriptional activation assays. Results Irx5 knock-out mice weighed less, had diminished fat mass, and were protected from diet-induced fat accumulation. Key adipose mitochondrial genes Pparγ coactivator 1-alpha (Pgc-1α) and uncoupling protein 1 (Ucp1) were upregulated, and a gene network centered on amyloid precursor protein (App) was downregulated in adipose tissue of knock-out mice and in isolated mouse adipocytes with stable Irx5 knock-down. An APP-centered network was also enriched in isolated adipocytes from obese compared to lean humans. IRX5 overexpression increased APP promoter activity and both IRX5 and APP inhibited transactivation of PGC-1α and UCP1. Knock-down of Irx5 or App increased mitochondrial respiration in adipocytes. Conclusion Irx5-KO mice were protected from obesity and this can partially be attributed to reduced adipose App and improved mitochondrial respiration. This novel Irx5-App pathway in adipose tissue is a possible therapeutic entry point against obesity.

Tong Liu,* Xin Zhang,* Yong-mei Yang, Lu-tao Du, Chuan-xin Wang Department of Clinical Laboratory, Qilu Hospital, Shandong University, Jinan, People’s Republic of China *These authors contributed equally tothis work Background: The long noncoding RNA (lncRNA) colorectal neoplasia differentially expressed– h (CRNDE-h) plays important roles in the early stages of human development and cancer progression. We investigated the expression and clinical significance of lncRNA CRNDE-h in colorectal cancer (CRC). Methods: The expression level of lncRNA CRNDE-h was analyzed in 142 CRC tissues and 142 paired adjacent nontumorous tissues, along with 21 inflammatory bowel diseases, 69 hyperplastic polyp, and 73 colorectal adenoma samples, using quantitative real-time polymerase chain reaction. The association between lncRNA CRNDE-h, and Iroquois homeobox protein 5 (IRX5) mRNA was examined in the same 142 CRC tissues. Results: We found that lncRNA CRNDE-h level was elevated in the CRC and adenoma groups compared with the other groups (all at P

Genome-wide association studies (GWAS) identified >90 loci containing genetic variants, many in intronic regions, associated with human obesity. Understanding how these variants regulate gene expression has been challenging. Claussnitzer et al. present a strategy for deciphering non-coding complex trait genetic associations which greatly advances their functional analysis. The strongest genetic association with risk to polygenic obesity are single-nucleotide variants in intron 1 and 2 of the FTO (fat mass and obesity associated) gene (Yang et al., 2012; Locke et al., 2015). However, of the 89 genetic variants in FTO intron 1 and 2, the causal variants, and their mechanistic underpinning have been elusive. A new report by Claussnitzer et al. identified a causal variant (rs1421085) and revealed its function in preadipocytes. Their study, published in the New England Journal of Medicine, provides evidence for the rs1421085 T to C single-nucleotide alteration to result in a cellular phenotype consistent with obesity in primary human adipocytes, including decreased mitochondrial energy generation and increased triglyceride accumulation (Claussnitzer et al., 2015). GWAS have identified >90 loci and thousands of single-nucleotide-polymorphisms (SNPs) associated with body mass index (BMI). The majority of these SNPs are in non-coding regions of the genome and therefore do not directly alter protein sequence which makes their interpretation challenging (Zhang et al., 2014). In fact, intronic GWAS signals, which characteristically encompass a large number of variants in linkage disequilibria, have very rarely lead to the discovery of the disease-causing variants or the mechanism by which they affect disease susceptibility. Interestingly, many GWAS regions are enriched for histone modifications (The ENCODE Project Consortium, 2012), suggesting they may play a role in gene regulation. Due to the time and tissue-specificity and the developmental and epigenetic complexity of gene regulation, functional approaches require the study of relevant human tissues and cells as well as bioinformatics approaches (e.g., the search for phylogenetic conservation) that reliably assess the regulatory role of non-coding variants (Claussnitzer et al., 2014). Using a range of bioinformatics and genetic approaches across diverse human tissues and cell types, Claussnitzer et al. unraveled the functional circuitry of the FTO locus. They present a model that deciphered: (1) the relevant tissue and cell type; (2) the regulated genes; (3) the causative variant; (4) the upstream regulator; (5) the cellular phenotype; and (6) the organismal phenotype (Figure ​(Figure1).1). Specifically, their study shows that intron 1 of FTO harbors a 10 kb potent super-enhancer which is active in the mesenchymal and adipocyte lineages. This enhancer region contains the variant rs1421085 in a highly conserved motif comprising a binding site for ARID5B. ARID5B when binding to this motif acts as a transcriptional repressor. The FTO risk allele disrupts binding of ARID5B thereby giving access to a super-enhancer active in preadipocytes, resulting in the over-expression of two genes distal to FTO, namely IRX3 and IRX5. Ultimately this leads to a cell-autonomous shift from mitochondrial energy production to lipid accumulation in risk allele carriers. Strikingly, genome editing of primary human preadipocyte samples toward the protective allele rescued the associated cellular phenotype, causing brown-like adaptation in the adipocyte as shown by up-regulation of thermogenesis. In their study, Claussnitzer et al. show that decreased expression of IRX3 and IRX5 in pre-adipocytes caused them to develop into brown-like adipocytes, with increased mitochondrial uncoupling which was maintained in mature adipocytes. Using the aP2-CRE for adipose-specific IRX3 inhibition, they show that at the organismal level, adipose specific IRX3 knockdown in mice resulted in 57% reduced fat mass ratio and high-fat-diet induced obesity resistance. This effect was significantly larger compared to the hypothalamus-specific IRX3 inhibition (Ins2-IRX3DN) which resulted in 19% reduced fat mass ratio. These compelling results suggest that the rs1421085 single-nucleotide change can alter metabolism that is responsible for the association between the first intron of FTO and obesity in humans. Further, this study clearly identifies adipocyte precursor cells as one of the primary effectors of FTO locus activity. Figure 1 Model for deciphering non-coding variants identified by GWAS after Claussnitzer et al. (2015). (A) Approach to study genes identified by GWAS involved establishment of relevant tissue and cell types, target genes, causative variants, upstream regulator, ... Claussnitzer et al. identify the causal variant in the FTO locus and reveal its function specifically in preadipocytes, where the risk allele affects the basic anabolic function of the adipocyte, shifting it from substrate storage to fuel dissipation through increased mitochondrial uncoupling. What remains to be determined is the precise molecular mechanism by which IRX3 and IRX5 influence mitochondrial energy consumption and metabolism on an organismal level. Mouse models of increased IRX3 and IRX5 expression as well as in vivo models harboring the human mutations are necessary to fully understand the association between FTO intron 1 and obesity. A further intriguing question is whether additional conserved motifs and SNPs in FTO, other than rs1421085, can be functionally linked to disturbed metabolism and whether other tissues or cell types might be affected (Smemo et al., 2014). The FTO gene itself has recently been shown to play a role specifically in adipocyte precursors where it influences early processes in the differentiation cascade (Zhao et al., 2014; Merkestein et al., 2015) and FTO-knockout (in which a part of Fto exon 3 is deleted) results in a similar adipose phenotype as the IRX3-knockout, raising the question of the role of FTO itself in this cluster of adipocyte metabolism genes. Demonstrated by their work on the FTO locus, Claussnitzer et al. present a strategy for deciphering non-coding complex trait genetic associations which can be used to functionally analyze GWAS hits in the future. In addition, this study very elegantly provides evidence for the clinical importance of mitochondrial activity and thermoregulation in white adipose tissue in obesity. By presenting a causative SNP, its regulator and a cellular phenotype, this study certainly provides new important insight and will guide future research into one of the most prominent obesity loci. Claussnitzer et al. show that FTO obesity risk variants cell-autonomously and dynamically modulate mitochondrial activity of human white adipose tissue, a cellular phenotype consistent with obesity. This finding raises the possibility of thermogenesis in adipose tissue as a therapeutic target. Importantly, this study provides a convincing model for translating information arising from GWAS.

In the mouse retina, at least ten distinct types of bipolar interneurons are involved in the transmission of visual signals from photoreceptors to ganglion cells. How bipolar interneuron diversity is generated during retinal development is poorly understood. Here, we show that Irx5, a member of the Iroquois homeobox gene family, is expressed in developing bipolar cells starting at postnatal day 5 and is localized to a subset of cone bipolar cells in the mature mouse retina. In Irx5-deficient mice, defects were observed in the expression of some, but not all, immunohistological markers that define mature Type 2 and Type 3 OFF cone bipolar cells, indicating a role for Irx5 in bipolar cell differentiation. The differentiation of these two bipolar cell types has previously been shown to require the homeodomain-CVC transcription factor, Vsx1. However, the defects observed in Irx5-deficient retinas do not coincide with a reduction of Vsx1 expression, and conversely, the expression of Irx5 in cone bipolar cells does not require the presence of a functional Vsx1 allele. These results indicate that there are at least two distinct genetic pathways (Irx5-dependent and Vsx1-dependent) regulating the development of Type 2 and Type 3 cone bipolar cells.

CRNDE is the gene symbol for Colorectal Neoplasia Differentially Expressed (non-protein-coding), a long non-coding RNA (lncRNA) gene that expresses multiple splice variants and displays a very tissue-specific pattern of expression. CRNDE was initially identified as a lncRNA whose expression is highly elevated in colorectal cancer, but it is also upregulated in many other solid tumors and in leukemias. Indeed, CRNDE is the most upregulated lncRNA in gliomas and here, as in other cancers, it is associated with a “stemness” signature. CRNDE is expressed in specific regions within the human and mouse brain; the mouse ortholog is high in induced pluripotent stem cells and increases further during neuronal differentiation. We suggest that CRNDE is a multifunctional lncRNA whose different splice forms provide specific functional scaffolds for regulatory complexes, such as the polycomb repressive complex 2 (PRC2) and CoREST chromatin-modifying complexes, which CRNDE helps pilot to target genes.