Human Macrophage Long Intergenic Noncoding RNA, SIMALR, Suppresses Inflammatory Macrophage Apoptosis via NTN1 (Netrin-1).

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as novel regulators of macrophage biology and inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human lncRNAs that are not conserved in rodents, are limited. METHODS: Through RNA-sequencing of human monocyte-derived macrophages, we identified suppressor of inflammatory macrophage apoptosis lncRNA (SIMALR). Lipopolysaccharide/IFNγ (interferon γ) stimulated human macrophages were treated with SIMALR antisense oligonucleotides and subjected to RNA-sequencing to investigate the function of SIMALR. Western blots, luciferase assay, and RNA immunoprecipitation were performed to validate function and potential mechanism of SIMALR. RNAscope was performed to identify SIMALR expression in human carotid atherosclerotic plaques. RESULTS: RNA-sequencing of human monocyte-derived macrophages identified SIMALR, a human macrophage-specific long intergenic noncoding RNA that is highly induced in lipopolysaccharide/IFNγ-stimulated macrophages. SIMALR knockdown in lipopolysaccharide/IFNγ stimulated THP1 human macrophages induced apoptosis of inflammatory macrophages, as shown by increased protein expression of cleaved PARP (poly[ADP-ribose] polymerase), caspase 9, caspase 3, and Annexin V+. RNA-sequencing of control versus SIMALR knockdown in lipopolysaccharide/IFNγ-stimulated macrophages showed Netrin-1 (NTN1) to be significantly decreased upon SIMALR knockdown. We confirmed that NTN1 knockdown in lipopolysaccharide/IFNγ-stimulated macrophages induced apoptosis. The SIMALR knockdown-induced apoptotic phenotype was rescued by adding recombinant NTN1. NTN1 promoter-luciferase reporter activity was increased in HEK293T (human embryonic kidney 293) cells treated with lentiviral overexpression of SIMALR. NTN1 promoter activity is known to require HIF1α (hypoxia-inducible factor 1 subunit alpha), and our studies suggest that SIMALR may interact with HIF1α to regulate NTN1 transcription, thereby regulating macrophages apoptosis. SIMALR was found to be expressed in macrophages in human carotid atherosclerotic plaques of symptomatic patients. CONCLUSIONS: SIMALR is a nonconserved, human macrophage lncRNA expressed in atherosclerosis that suppresses macrophage apoptosis. SIMALR partners with HIF1α (hypoxia-inducible factor 1 subunit alpha) to regulate NTN1, which is a known macrophage survival factor. This work illustrates the importance of interrogating the functions of human lncRNAs and exploring their translational and therapeutic potential in human atherosclerosis.

Cis-regulated expression of non-conserved lincRNAs associates with cardiometabolic related traits.

Many complex disease risk loci map to intergenic regions containing long intergenic noncoding RNAs (lincRNAs). The majority of these is not conserved outside humans, raising the question whether genetically regulated expression of non-conserved and conserved lincRNAs has similar rates of association with complex traits. Here we leveraged data from the Genotype-Tissue Expression (GTEx) project and multiple public genome-wide association study (GWAS) resources. Using an established transcriptome-wide association study (TWAS) tool, FUSION, we interrogated the associations between cis-regulated expression of lincRNAs and multiple cardiometabolic traits. We found that cis-regulated expression of non-conserved lincRNAs had a strikingly similar trend of association with complex cardiometabolic traits as conserved lincRNAs. This finding challenges the conventional notion of conservation that has led to prioritization of conserved loci for functional studies and calls attention to the need to develop comprehensive strategies to study the large number of non-conserved human lincRNAs that may contribute to human disease.

Nonconserved Long Intergenic Noncoding RNAs Associate With Complex Cardiometabolic Disease Traits.

OBJECTIVE: Transcriptome profiling of human tissues has revealed thousands of long intergenic noncoding RNAs (lincRNAs) at loci identified through large-scale genome-wide studies for complex cardiometabolic traits. This raises the question of whether genetic variation at nonconserved lincRNAs has any systematic association with complex disease, and if so, how different this pattern is from conserved lincRNAs. We evaluated whether the associations between nonconserved lincRNAs and 8 complex cardiometabolic traits resemble or differ from the pattern of association for conserved lincRNAs. Approach and Results: Our investigation of over 7000 lincRNA annotations from GENCODE Release 33-GRCh38.p13 for complex trait genetic associations leveraged several large, established meta-analyses genome-wide association study summary data resources, including GIANT (Genetic Investigation of Anthropometric Traits), UK Biobank, GLGC (Global Lipids Genetics Consortium), Cardiogram (Coronary Artery Disease Genome Wide Replication and Meta-Analysis), and DIAGRAM (Diabetes Genetics Replication and Meta-Analysis)/DIAMANTE (Diabetes Meta-Analysis of Trans-Ethnic Association Studies). These analyses revealed that (1) nonconserved lincRNAs associate with a range of cardiometabolic traits at a rate that is generally consistent with conserved lincRNAs; (2) these findings persist across different definitions of conservation; and (3) overall across all cardiometabolic traits, approximately one-third of genome-wide association study-associated lincRNAs are nonconserved, and this increases to about two-thirds using a more stringent definition of conservation. CONCLUSIONS: These findings suggest that the traditional notion of conservation driving prioritization for functional and translational follow-up of complex cardiometabolic genomic discoveries may need to be revised in the context of the abundance of nonconserved long noncoding RNAs in the human genome and their apparent predilection to associate with complex cardiometabolic traits.

linc-ADAIN, a human adipose lincRNA, regulates adipogenesis by modulating KLF5 and IL-8 mRNA stability.

Adipose tissue remodeling and dysfunction, characterized by elevated inflammation and insulin resistance, play a central role in obesity-related development of type 2 diabetes (T2D) and cardiovascular diseases. Long intergenic non-coding RNAs (lincRNAs) are important regulators of cellular functions. Here, we describe the functions of linc-ADAIN (adipose anti-inflammatory), an adipose lincRNA that is downregulated in white adipose tissue of obese humans. We demonstrate that linc-ADAIN knockdown (KD) increases KLF5 and interleukin-8 (IL-8) mRNA stability and translation by interacting with IGF2BP2. Upregulation of KLF5 and IL-8, via linc-ADAIN KD, leads to an enhanced adipogenic program and adipose tissue inflammation, mirroring the obese state, in vitro and in vivo. KD of linc-ADAIN in human adipose stromal cell (ASC) hTERT adipocytes implanted into mice increases adipocyte size and macrophage infiltration compared to implanted control adipocytes, mimicking hallmark features of obesity-induced adipose tissue remodeling. linc-ADAIN is an anti-inflammatory lincRNA that limits adipose tissue expansion and lipid storage.

Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity.

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.