Novel Lipid Long Intervening Noncoding RNA, Oligodendrocyte Maturation-Associated Long Intergenic Noncoding RNA, Regulates the Liver Steatosis Gene Stearoyl-Coenzyme A Desaturase As an Enhancer RNA

Abstract

The global obesity epidemic is driving the concomitant rise in nonalcoholic fatty liver disease (NAFLD). To identify new genes involved in central liver functions, we examined liver RNA-sequence data from 259 patients who underwent morbidly obese bariatric surgery. Of these patients, 84 had normal liver histology, 40 simple steatosis, 43 nonalcoholic steatohepatitis, and the remaining 92 patients had varying degrees of NAFLD based on liver histology. We discovered oligodendrocyte maturation-associated long intergenic noncoding RNA (OLMALINC), a long intervening noncoding RNA (lincRNA) in a human liver co-expression network (n = 75 genes) that was strongly associated with statin use and serum triglycerides (TGs). OLMALINC liver expression was highly correlated with the expression of known cholesterol biosynthesis genes and stearoyl-coenzyme A desaturase (SCD). SCD is the rate-limiting enzyme in monounsaturated fatty acids and a key TG gene that is known to be up-regulated in liver steatosis and NAFLD and resides adjacent to OLMALINC on the human chromosome 10q24.31. Next, we functionally demonstrated that OLMALINC regulates SCD as an enhancer-RNA (eRNA), thus describing the first lincRNA that functions as an eRNA to regulate lipid metabolism. Specifically, we show that OLMALINC promotes liver expression of SCD in cis through regional chromosomal DNA-DNA looping interactions. Conclusion: The primate-specific lincRNA OLMALINC is a novel epigenetic regulator of the key TG and NAFLD gene SCD.

Figure 1

Liver weighted gene co‐expression network analyses (WGCNA) identify a statin‐associated network module (i.e. the light cyan module). (A) The association results between the liver WGCNA modules and statin use, metabolic traits, and histologic liver phenotypes in the Finnish KOBS cohort. D1 indicates the aggregated meta‐liver trait for NAFLD (see Participants and Methods). Numbers in the cells and parentheses indicate effect sizes and FDRs, respectively. (B) Genes in the light cyan module (n = 75) are strongly associated with statin medication and involved in cholesterol synthesis. The strength of association with statin medication is highly correlated with the module membership of the light cyan module. The red line indicates the threshold for the Bonferroni‐corrected P value of 0.05. Abbreviations: ALT, alanine aminotransferase; DM, diabetes mellitus; HDL, high‐density lipoprotein; LDL, low‐density lipoprotein; WGCNA, weighted gene co‐expression network analysis.

Figure 2

OLMALINC resides downstream of SCD and demonstrates similar regulatory regions. (A) The annotated OLMALINC promoter (red) and enhancer (orange) demonstrate histone methylation marks, 5′ CAGE, and polymerase II ChIP‐seq binding sites using ENCODE data. There are two TSSs: the orange arrow denotes the enhancer‐TSS, while the red arrow highlights the promoter‐TSS. Our GRO‐seq data in HepG2 cells show active transcription and nascent OLMALINC RNA expression bidirectionally. (B) OLMALINC has SREBP1/2, pravastatin (pravastatin‐treated HepG2 cells with SREBP1/2 peaks), and RXRA binding sites where an LXRE (LXRE‐DR4) is identified using sequence comparisons. Abbreviations: CAGE, capped analysis of gene expression; RXRA, retinoid X receptor alpha.

Figure 3

OLMALINC expression is responsive to sterols, statins, and LXR agonists in HepG2 cells. (A) OLMALINC and SCD increase expression by RT‐qPCR in a time‐dependent manner under sterol‐depleted conditions supplemented with statin treatment (5% lipoprotein‐deficient media with 5 µM simvastatin and 50 µM mavelonic acid) when compared to sterol‐rich conditions (10% FBS) supplemented with DMSO vehicle control, similarly to SREBP2 and its downstream gene HMGCS1. Each time point was normalized to its DMSO 10% FBS‐treated time point. (B) OLMALINC gene expression increases after 24‐hour treatment of GW3695 (an LXRα and LXRβ agonist) when compared to the DMSO vehicle control in 5% LPDS with 5 µM simvastatin and 50 µM mavelonic acid, as measured by RT‐qPCR. Values are mean ± SD (n = 3) for A and C or mean ± SEM for B (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student t test was used for two groups). Abbreviations: DMSO, dimethyl sulfoxide; HMGCS1, 3‐hydroxy‐3‐methylglutaryl‐coenzyme A synthase 1.

Figure 4

OLMALINC ASO introduced to HepG2 cells causes a decrease in expression of OLMALINC and target genes. (A) OLMALINC and target gene expression, measured by RT‐qPCR, decrease after 24‐hour and 36‐hour treatment with ASO targeting exon 2 of the OLMALINC gene. (B) Validation of SCD protein antibody (38 kDa) after treatment with scramble, SCD, and SREBP1 with SREBP2 siRNAs after 96 hours. (C) OLMALINC gene expression increases after 48‐hour treatment with an SCD siRNA compared to the scramble control. Values are mean ± SD (n = 3). *P < 0.05, ***P < 0.001 (unpaired Student t test was used for two groups).

Figure 5

OLMALINC regulates SCD gene expression in cis by forming DNA–DNA looping interactions. (A) Promoter Capture Hi‐C data in HepG2 cells demonstrate DNA–DNA looping interactions between the OLMALINC enhancer/promoter and the SCD promoter/enhancer regions. (B) Endogenous OLMALINC overexpression using aCRISPR‐dCa9 gene editing increases expression of SCD. (C) Overexpression of the spliced OLMALINC stable transcript (exons 1‐3) for 48 hours does not affect SCD gene expression. Expression data are normalized to a GFP negative control. Values are mean ± SD (n = 3). **P < 0.01, ***P < 0.001 (unpaired Student t test was used for two groups). Abbreviations: GFP, green fluorescent protein; ns, not significant.

Figure 6

OLMALINC enhancer/promoter deletion using CRISPR‐Cas9 gene editing decreases SCD gene expression. (A) Schematic of primer designs for genomic PCR amplification of wild type versus CRISPR‐Cas9‐mediated OLMALINC promoter/enhancer deletion. Per ENCODE HepG2 chromatin state data, red highlights OLMALINC promoter while yellow highlights the enhancer. (B) Gel electrophoresis of PCR products from amplification of the wild type and CRISPR‐Cas9 OLMALINC enhancer/promoter deletions from the genomic DNA from HepG2 cells. (C) Evaluation of transfection efficiency of HepG2 with fluorescently labeled tracRNA with ATTO‐550 after 24 hours; left panel demonstrating bright field cells and right panel the corresponding labeled cells. (D) OLMALINC and SCD gene expression by RT‐qPCR after 48‐hour transfection with the Cas9 enzyme and OLMALINC gRNAs flanking the enhancer/promoter region. Values are mean ± SD (n = 3). **P < 0.01, ***P < 0.001 (unpaired Student t test was used for two groups). Abbreviations: bp, base pair; tracRNA, trans‐activating RNA.

Figure 7

OLMALINC is regulated by MUFAs but not by SREBP1/2. (A) SREBP1a, SREBP1c, and SREBP2 gene expression after SREBP1 and SREBP2 siRNA cotransfection for 48 hours, relative to scramble siRNA control. (B) OLMALINC expression does not decrease after a 48‐hour cotransfection with SREBP1 and SREBP2 siRNAs; SCD decreases. (C‐E) SREBP1a, SREBP1c, and SCD expression decreases after lipid loading with MUFAs (200 µM oleic acid) 24‐hour treatment only, following 8 hours of starvation in 0.5% FBS. (F) OLMALINC decreases its expression after lipid loading with MUFAs (200 µM oleic acid) after 18‐hour and 24‐hour treatment, following 8 hours of starvation in 0.5% FBS. All expression time points are normalized to the corresponding gene expression in 0.5% FBS. Values are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student t test was used for two groups).