GSE212381
Human Macrophage Long Intergenic Non-coding RNA, SIMALR, Suppresses Inflammatory Macrophage Apoptosis via Netrin 1
Long noncoding RNAs (lncRNAs) have emerged as novel regulators of macrophage biology and related inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human lncRNAs that are not conserved in rodents, are limited. Through RNA-seq of human monocyte-derived macrophages, we identified Suppressor of Inflammatory Macrophage Apoptosis lncRNA (SIMALR), a human macrophage-specific long intergenic noncoding RNA, to be highly induced in LPS/IFN?-stimulated macrophages. Treatment of LPS/IFN? stimulated THP1 human macrophages with SIMALR antisense oligonucleotides induced apoptosis of inflammatory macrophages, as shown by increased protein expression of cleaved PARP, caspase 9, caspase 3, and Annexin V+. RNA-seq of control versus SIMALR knockdown in LPS/IFN?-stimulated human monocyte derived macrophages showed NTN1 to be significantly decreased upon SIMALR knockdown. As expected, NTN1 knockdown in LPS/IFN?-stimulated macrophages induced apoptosis. This apoptotic phenotype was attenuated by adding recombinant NTN1 after SIMALR knockdown. Furthermore, NTN1 promoter-luciferase reporter activity was increased in HEK293T cells treated with lentiviral overexpression of SIMALR. NTN1 promoter activity is known to require HIF1a. RNA immunoprecipitation and microscopy studies suggest that SIMALR may interact with HIF1a to regulate NTN1 transcription, thereby regulating apoptosis of macrophages. In translational studies, SIMALR was found to be expressed in macrophages in human carotid atherosclerotic plaques of symptomatic patients. SIMALR is a non-conserved, human macrophage lincRNA expressed in human atherosclerosis that regulates macrophage apoptosis.  SIMALR partners with HIF1a to regulate NTN1, which is a known macrophage survival factor. This work illustrates the importance of interrogating the functions of non-conserved human lincRNAs and exploring their translational and therapeutic potential in human atherosclerosis. Overall design: RNA-seq was performed on LPS/IFN?-stimulated human monocyte-derived macrophages that were treated with SIMALR antisense oligonucleotides or control. Ingenuity Pathway Analysis (IPA) was next performed on differentially expressed genes identified from RNA-seq. IPA predicted that SIMALR may play a role in macrophage apoptosis. To verify this prediction, we performed Annexin V+ flow analysis and western blot with apoptotic markers on LPS/IFN? stimulated THP1 human macrophages after SIMALR knockdown. RNA-seq data also revealed NTN1, a known macrophage survival marker, to be significantly downregulated with SIMALR knockdown. To test whether NTN1 expression was regulated by SIMALR, luciferase assay with NTN1 promoter-luciferase reporter was performed after treating HEK293T cells with lentiviral overexpression of SIMALR. As NTN1 is known to be regulated by HIF1a, we also investigated whether SIMALR interacts with HIF1a with RNA immunoprecipitation and microscopy studies in LPS/IFN? stimulated THP1 human macrophages. Finally, to see if SIMALR can be detected in human atherosclerotic plaques, we performed RNAscope assay to probe for SIMALR, along with CD68 and ACTA2.