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. 2020 Jul:37:100996.
doi: 10.1016/j.molmet.2020.100996. Epub 2020 Apr 14.

The novel long noncoding RNA Lnc19959.2 modulates triglyceride metabolism-associated genes through the interaction with Purb and hnRNPA2B1

Jing Wang  1 , Dao Xiang  1 , Shuai Mei  1 , Yuanchao Jin  1 , Dating Sun  1 , Chen Chen  2 , Dong Hu  1 , Shiyang Li  1 , Huihui Li  1 , Yan Wang  2 , Dao Wen Wang  3 , Hu Ding  4
Affiliations

The novel long noncoding RNA Lnc19959.2 modulates triglyceride metabolism-associated genes through the interaction with Purb and hnRNPA2B1

Jing Wang et al. Mol Metab. 2020 Jul.

Abstract

Objective: Long noncoding RNAs (lncRNAs) are currently considered to have a vital and wide range of biological functions, but the molecular mechanism underlying triglycerides metabolism remains poorly understood. This study aims to identify novel lncRNAs differentially expressed in rat livers with hypertriglyceridemia and elucidated the function role in TG metabolism.

Methods: Differentially expressions of lncRNAs in rat livers with hypertriglyceridemia were identified by transcriptome sequencing and validated by real-time PCR. The role of lnc19959.2 in triglyceride metabolism was assessed both in vitro and in vivo. RNA pulldown and RIP assays were conducted to evaluate the interactions between lnc19959.2 and its target proteins. ChIP and Dual report assays were performed to detect the interactions between transcription factors and promoters of its target genes.

Results: We identified a novel lncRNA, and lnc19959.2 was upregulated in rat livers with hypertriglyceridemia. The knockdown of lnc19959.2 has profound TG lowering effects in vitro and in vivo. Subsequently, the genome-wide analysis identified that the knockdown of lnc19959.2 caused the deregulation of many genes during TG homeostasis. Further mechanism studies revealed that lnc19959.2 upregulated ApoA4 expression via ubiquitinated transcription inhibitor factor Purb, while it specifically interacted with hnRNPA2B1 to downregulate the expression of Cpt1a, Tm7sf2, and Gpam, respectively. In the upstream pathway, palmitate acid upregulated CCAAT/Enhancer-Binding Protein Beta (Cebpb) and facilitated its binding to the promoter of lnc19959.2, which resulted in significant promotion of lnc19959.2 transcriptional activity.

Conclusions: Our findings provide novel insights into a new layer regulatory complexity of an lncRNA modulating triglyceride homeostasis by a novel lncRNA lnc19959.2.

Keywords: Epigenetics; LncRNAs; Transcriptional regulation; Triglyceride metabolism.

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Figures

Image 1
Graphical abstract
Figure 1
Figure 1
Lnc19959.2 is a novel lncRNA highly expressed in rat fatty livers. (A) Schematic diagram shows rat livers (ND, n = 3; HFD, n = 5) selected for RNA-seq. (B) Serum total triglyceride (TG) levels in normal diet (ND) and high-fat diet (HFD) rats. Serum TG was significantly increased after 8 weeks in HFD rats. (C) Heatmap of 832 different expression lncRNAs (ND, n = 3; HFD, n = 5). (D) The number of significantly changed lncRNAs (red indicates the upregulated lncRNAs, with fold change > 2.0; green indicates the downregulated lncRNAs, with fold change<0.5). (E) RT-qPCR analysis the lncRNA expressions after treating BRL-3A cells with palmitate acid (300 μM) for 24 h. The CT value of GAPDH was 13.483 ± 1.192 (n = 3) and lnc19959.2 was 21.791 ± 0.171 (n = 3, mean ± SD). Unpaired t-test was used to measure the statistical significance; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. Results are mean ± SEM of three independent experiments with duplicate wells.
Figure 2
Figure 2
Knockdown lnc19959.2 reduces plasma TG levels in rats. (A) RT-qPCR analysis lnc19959.2 expression level following transient transfection of BRL-3A cells with smart siRNA of lnc19959.2. (B–C) The TC and TG levels detected in BRL-3A cells after transient transfection siRNA of lnc19959.2 with or without PA. (D–E) Oil Red O-hematoxylin staining of BRL-3A cells after transient transfection siRNA of lnc19959.2 with PA. (F) Plasma TG levels in high-fat diet (HFD) rats of control (GFP, n = 6) and lnc19959.2 KD (KD-lnc19959.2, n = 9) after a 6-hour fast (Fast 6 h), a 24-hour fast (Fast 24 h), or 12-hour refeeding after a 24-hour fast (Refeed 12 h). (G) Blood triglyceride levels detected at different time points (0, 0.5, 1, 2, and 3 h) after receiving olive oil by oral gavage in ND rats which are injected with adenovirus and, for another group, after receiving olive oil and 0.9% sodium chloride, separately (∗P < 0.05 means 0.9% sodium chloride versus olive oil, and P < 0.05 means GFP versus KD-lnc19959.2). Unpaired t-test was used to measure the statistical significance; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.
Figure 3
Figure 3
lnc19959.2 participates in many metabolism pathways and affects a series of TG-associated genes. (A) Heatmap of mRNA expression profile sequence, after knockdown of lnc19959.2 expressions in BRL-3A cells. (B) Gene ontology analysis of microarray in BRL-3A cell treatment of siRNA of lnc19959.2 and Negative Control (NC). (C) The top 30 KEGG pathways correlated with the cellular process, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems. (D) RT-qPCR detected a subset of the lipid-related pathway gene expressions.
Figure 4
Figure 4
lnc19959.2 interacts with Purb and hnRNPA2B1. (A) Experimental design for RNA pulldown assay to identify the cellular proteins associating with lnc19959.2. lnc19959.2 was transcription-labeled with biotin in vitro, refolded, and incubated with BRL-3A total cell lysates. (B) Venn diagram showing the number of different proteins binding with lnc19959.2_sense and lnc19959.2_antisense, separately, according to the MS analysis. And 10 proteins were overlapped. (C) Four of the candidate proteins filtered from mass spectrometry. Purb and hnRNPA2B1 were confirmed that they were specifically bound to lnc19959.2. (D) RIP assay analysis of Purb and hnRNPA2B1 associated with lnc19959.2. BRL-3A cell lysates were immunoprecipitated with anti-Purb and anti-hnRNPA2B1 or control mouse IgG antibody. RT-qPCR was used to validate the presence of lnc19959.2 in the complexes. (E) The total length of lnc19959.2 minimum free energy (MFE) secondary structure. This structure is colored by base-pairing probabilities. And red shows high confidence and blue is just the opposite. (F) The different length (full-length (FL) and truncated lnc19959.2 (Δ1: 1–650, Δ2: 1–1300, Δ3: 1–1950, FL: 1–2501, Δ4: 651–1300, Δ5: 1301–1950, Δ6: 1951–2501)) of lnc19959.2 were biotinylated in RNA pulldown assays and western blot was used to detect Purb and hnRNPA2B1. Unpaired t-test was used to measure the statistical significance; ∗∗∗∗P < 0.0001.
Figure 5
Figure 5
Purb acts as a transcriptional repressor to inhibit ApoA4 expression. (A) Western blot detected the ApoA4 and Purb expression levels after knockdown or overexpression of Purb in BRL-3A cells. (B) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of four truncated ApoA4 promoters with pcDNA3.1 vector or pc3.1-Purb in HEK293T cells. (C) The schematic diagram shows that Purb binding site (PNR) contained at 2000 bp of ApoA4 promoter was well conserved. (D) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of ApoA4 promoters of 2000 bp with pcDNA3.1 vector or pc3.1-Purb in different doses (range: 0–1.5 μg/mL) in HEK293T cells. (E) Dual-luciferase reporter assay detected relative luciferase activities after cotransfected with wt/mutApoA4 promoters of 2000 bp with pc3.1-Purb in HEK293T cells. (F) ChIP analysis of Purb interaction with the ApoA4 promoter. BRL-3A cells lysates were immunoprecipitated with anti-Purb or control mouse IgG antibody. (G–H) Overexpression of lnc19959.2 and control in BRL-3A cells, respectively, were incubated with the proteasome inhibitor MG-132 (10 μM) or the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) for 6 or 12 h. The protein level of Purb extracted from the whole cell was detected by western blot. (I–J) BRL-3A cells lysates were immunoprecipitated with an HA or Flag-specific antibody in BRL-3A cell lysates which were stably expressing ubiquitin with C-terminal HA tag or Purb with C-terminal Flag tag, respectively. And then they were analyzed by western blot with anti-Purb or anti-Ubiquitin. Bottom, the input of the cell lysates. (K) Western blot detects the ubiquitination levels in BRL-3A cells. Unpaired t-test was used to measure the statistical significance; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
Figure 6
Figure 6
Cpt1a, Tm7sf2, and Gpam are coregulatory by lnc19959.2-hnRNPA2B1. (A–B) The top 30 gene ontology (GO) related to biological process (BP), cellular component (CC), and molecular function (MF) and the top 30 KEGG pathways correlated with the cellular process, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems in hnRNPA2B1 knockdown BRL-3A cells. (C) ChIP-qPCR analysis recruitment of hnRNPA2B1 to promoter regions determined in BRL-3A cells transduced with control (pcDNA3.1) or lnc19959.2 overexpression (pc3.1-lnc19959.2). The results retrieved and normalized by an upstream site (region 1) are shown as a percent of input (n = 3 per group). (D) ChIP-qPCR analysis recruitment of RNA polymerase II (RNPII) to promoter regions determined in BRL-3A cells transduced with control (pcDNA3.1) or lnc19959.2 overexpressing plasmid (pc3.1-lnc19959.2). Results retrieved and normalized by an upstream site (region1) are shown as a percent of input (n = 3 per group). (E) Western blot detected Cpt1a, Tm7sf2, and Gpam protein expression levels. Unpaired t-test was used to measure the statistical significance; ∗∗P < 0.01.
Figure 7
Figure 7
Cebpb activates the lnc19959.2 expression. (A) Heatmaps present gene expressions selected from sequence prediction software. (B–C) RT-qPCR analysis of lnc19959.2 expression levels in BRL-3A cells in the condition of knockdown or overexpression of Cebpb. (D) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of four truncated lnc19959.2 promoters with pcDNA3.1 vector or pc3.1-Cebpb in HEK293T cells. (E) Dual-luciferase reporter assay detected relative luciferase activities after cotransfection of lnc19959.2 promoters of 1500 bp with pcDNA3.1vector or pc3.1-Cebpb in different doses (range: 0–1.5 μg/mL) in HEK293T cells. (F) Schematic diagram shows that the part of lnc19959.2 promoter sequence of wt and mut of Cebpb binding site is located in −1500～-1000 bp. (G) Dual-luciferase reporter assay detected relative luciferase activities after being cotransfected with wt/mutlnc19959.2 promoters of 1500 bp with pc3.1-Cebpb in HEK293T cells. (H) ChIP analysis of Cebpb interacted with the lnc19959.2 promoter. BRL-3A cells lysates were immunoprecipitated with anti-Cebpb or control mouse IgG antibody. Unpaired t-test was used to measure the statistical significance; ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.
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