Research ArticleAtherosclerosis

Long noncoding RNA SNHG12 integrates a DNA-PK–mediated DNA damage response and vascular senescence

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Science Translational Medicine  19 Feb 2020:
Vol. 12, Issue 531, eaaw1868
DOI: 10.1126/scitranslmed.aaw1868
  • Fig. 1 Identification of the conserved lncRNA SNHG12 in lesional intima.

    (A) RNA derived from aortic intima of Ldlr−/− mice (n = 3; each sample represents RNA pooled from two mice) placed on HCD for 0 weeks (group 1), 2 weeks (group 2), 12 weeks (group 3), or 18 weeks (group 4) after 6 weeks of resumption of a normal chow diet. (B) Workflow of genome-wide RNA-seq profiling for the identification of differentially expressed lncRNAs [log2 fold change (FC), >1.5; FDR, <0.05]. (C) Venn diagram displaying dysregulated lncRNAs detected by DESeq2 with or without overlapping reads. (D) 5′RACE-PCR for Snhg12 in mouse from RNA of the aortic intima and human RNA from HUVECs (n = 3). Visualization of RNA-seq in mouse and pig to verify sequence alignment and splicing junctions. (E) RNA-seq of Snhg12 in groups 1 to 4 was verified by RT-qPCR for the Snhg12-205 isoform. (F) Ldlr−/− mice were intravenously injected with vehicle control–gapmeR or SNHG12-gapmeR (7.5 mg/kg per mouse) twice per week and placed on HCD for 12 weeks (n = 12 per group) and assessed for (G) Snhg12 knockdown (KD) in aortic intima, media, and PBMCs (n = 6 per group). (H) Lesion areas were detected by Oil Red O (ORO) staining aortic sinus sections (n = 10 per group) and (I) thoracoabdominal aorta (n = 12 per group). (J) ApoE−/− mice were intravenously injected with LacZ or Snhg12 RNA twice per week and placed on an HCD for 6 weeks (n = 10 per group). (K) Delivery of RNA to the aortic intima, tunica media, and PBMCs was assessed by RT-qPCR (n = 5 per group). Lesion areas were quantified by Oil Red O staining of aortic sinus (L) and thoracoabdominal aorta (M) (n = 10 per group). All P values by Student’s t test. For all panels, values are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 LncRNA SNHG12 interacts with DNA-PK.

    (A) Proteins identified in DNA-PK and negative control LacZ pulldowns of biotinylated SNHG12 (n = 2 independent biological experiments, n = 2 technical replicates each). (B) Immunoblotting for DNA-PK, ATM, ATR, Ku80, Ku70, p53, and control on nuclear protein lysate from HUVECs after lncRNA pulldown (n = 5). (C) Western blot after streptavidin pulldown of nuclear protein lysate derived from the aorta of C57Bl/6 mice after two intravenous injections of biotin-labeled Snhg12 or LacZ (n = 4 per group). (D) Predicted secondary structure of SNHG12 with indicated deletions of domains 1 to 4. (E) LncRNA pulldown of biotinylated SNHG12 domain deletion constructs (n = 4). P value by one-way ANOVA with Fisher’s test. (F) Immunoprecipitation of DNA-PK after RNA isolation and subsequent RT-qPCR for SNHG12 and for negative control HPRT (n = 5). (G) HUVEC nuclear protein lysate was harvested 36 hours after transfection with control-gapmeR or SNHG12-gapmeR (25 nM) in the presence or absence of H2O2 for 1 hour (1 mM), immunoprecipitated with IgG or DNA-PK antibody, and immunoblotted for Ku70, Ku80, and DNA-PK. (H) Quantification of (G) under H2O2 conditions (n = 3). For all panels, values are means ± SD; **P < 0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; n.d., not detectable; WT, wild type; IgG, immunoglobulin G; USF-2, upstream stimulatory factor 2.

  • Fig. 3 DNA damage analysis in vitro and in lesions upon SNHG12 loss- and gain-of-function studies.

    (A) SNHG12 expression was analyzed in HUVECs γ-irradiated for 1 min (1.2 Gy−min) and RNA isolated at 0, 1, 2, 4, 8, or 12 hours after irradiation or in the absence or presence of H2O2 (30, 100, 250, 500, or 1000 μM) for 4 hours. (B) Control-gapmeR– or SNHG12-gapmeR–transfected HUVECs were γ-irradiated (1.2 Gy−min) and fixed at 0, 1, 2, 4, 8, or 12 hours after irradiation. Quantification of γH2AX foci with representative images. Scale bar, 20 μm. IR, irradiation. (C) Western blot analysis of protein lysate for γH2AX from HUVECs transfected with gapmeRs or negative control treated for 0 and 30 min with H2O2 (1 mM) (n = 3). (D) HUVECs transduced with control lentivirus or SNHG12 lentivirus were analyzed for γH2AX in the absence or presence of H2O2 (1 mM) for 1 hour. (E) Lesional DNA damage in the vessel wall of the aortic arch in Ldlr−/− mice on HCD injected with control-gapmeR or SNHG12-gapmeR for 12 weeks was quantified by γH2AX with nuclear colocalization of DAPI in lesional CD31+ cells. Representative images are shown. Scale bar, 50 μm (n = 6 mice per group; two to three lesions per arch). (F) Lesional DNA damage was quantified in the aortic arch of ApoE−/− mice after delivery of lacZ or Snhg12 RNA. Representative images are shown. Scale bar, 200 μm (n = 10 mice per group). (G and H) HUVECs were cotransfected with either control-gapmeR, SNHG12-gapmeR, control-siRNA, or siRNA–DNA-PK (25 nM each) and treated with H2O2 (1 mM) for 1 hour before DNA double-strand breaks (DSBs) were assessed by (G) γH2AX Western blot (n = 3) and (H) Comet assay (neutral pH) (n = 3). (I) NHEJ efficiency in HUVECs assessed by FACS for GFP-positive cells as an indicator or repaired DSB in HUVECs overexpressing SNHG12 (cherry reporter) compared to lentiviral control (n = 3). All P values by Student’s t test except for one-way ANOVA with Fisher’s test in (A). For all panels, values are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 Downstream consequences of SNHG12 on p53 and senescence.

    (A) Genome-wide RNA-seq profiling of HUVECs transfected with control-gapmeR or SNHG12-gapmeR treated for 1 hour with H2O2 (1 mM) (n = 3 per group). Volcano plot displaying significantly dysregulated genes (log2 fold change, >1.5; FDR, <0.05). (B) GSEA of the top 10 significantly affected processes and (C) enrichment plot for the p53 pathway of cells in (A) after SNHG12 knockdown. “Tag%” gives an indication of the fraction of genes contributing to the enrichment score. TNF, tumor necrosis factor. (D) p-p53 and total p53 by Western blot in gapmeR-transfected HUVECs with and without H2O2. P values by Student’s t test. (E) Electrophoretic mobility shift assay for p53 using nuclear lysate of control- or SNHG12-gapmeR–transfected HUVECs. (F) p16 and p21 immunoblot in cells treated for 1 hour with H2O2 (30 μM), followed by 3 days of incubation in normal growth medium. (G) RT-qPCR of senescence markers in mouse aortic endothelium–derived RNA (n = 6 per group). (H) Plaque necrosis in the aortic sinus (n = 6 per group; two to three lesions per mouse). Scale bar, 50 μm. (I) βgal staining of lentiviral SNHG12-transduced HUVECs. Representative images are shown. Scale bar, 300 μm (n = 3). (J) Transcytosis of DiI-labeled LDL quantified by TIRF microscopy (movies S1 and S2) (n = 3). (K) In vivo efferocytosis measured by Mac2-associated TUNEL staining in lesions of SNHG12-gapmeR–injected Ldlr−/− mice. Representative images are shown. Scale bar, 100 μm. P value by Student’s t test. For all panels, values are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 NR rescued progression of atherosclerotic lesions in Ldlr −/− mice induced by Snhg12 silencing.

    (A) Ldlr−/− mice were intravenously injected with vehicle control or SNHG12-gapmeR (7.5 mg/kg per mouse) twice per week and placed on an HCD containing NR (400 mg/kg per day) for 12 weeks (n = 12 per group). (B) Lesion areas were quantified using Oil Red O area on mouse aortic sinus sections (n = 10 per group). Representative images are shown. Scale bars, 200 μm. Fold change was calculated to no NR–treated, control-gapmeR–injected mice. (C) Lesional DNA damage in the vascular endothelium of the aortic arch was quantified in Ldlr−/− mice by γH2AX alongside nuclear colocalization with DAPI in CD31+ cells. Representative images are shown. Scale bar, 100 μm. RT-qPCR of (D) Snhg12 and (E) senescence markers p16, p21, and p27 from RNA isolated from the aortic intima of mice on HCD plus NR (n = 6 per group). (F) Acellular areas in the aortic sinus for each of the indicated groups (n = 6 per group; two to three lesions per mice). Representative images shown are shown. (G) TUNEL staining was quantified in the aortic sinus for each of the indicated groups (n = 7 per group). All P values by Student’s t test. For all panels, values are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 6 SNHG12 expression inversely correlates with DNA damage and senescence markers in human and pig atherosclerotic specimens.

    (A) SNHG12 expression in RNA isolated from nondiseased control human carotid arteries (n = 8) or atherosclerotic carotid arteries (n = 23). P value by Student’s t test. (B) Total γH2AX, p16, and p21 protein assessed from the same samples in (A), normalized by GAPDH. P value by Student’s t test. Plots in (A) and (B) indicate fold change relative to control arteries. (C) Oil Red O staining of fresh-frozen carotid artery cross sections from Yorkshire pigs fed an HCD for up to 60 weeks. (D) RT-qPCR analysis of SNHG12 expression in RNA from specimens in (C), normalized by GAPDH. P value by one-way ANOVA with Fisher’s test. (E) RNA-seq transcriptomic analysis of p16 and p21 expression. P value by one-way ANOVA with Fisher’s test. For all panels, values are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/531/eaaw1868/DC1

    Materials and Methods

    Fig. S1. Identification and characterization of lncRNA SNHG12 in mouse and human cells.

    Fig. S2. SNHG12 does not affect lipid metabolism or inflammation.

    Fig. S3. Identification of DNA-PK as an SNHG12 interactor.

    Fig. S4. SNHG12 silencing impairs DDR in ECs and macrophages.

    Fig. S5. Phenotypic effects of SNHG12 on senescence, EC permeability, and efferocytosis.

    Fig. S6. SNHG12 has no regulatory role in apoptosis.

    Fig. S7. Accumulating DNA damage and its effect on mitochondrial stress.

    Fig. S8. Proposed mechanism of SNHG12 regulation of atherosclerosis through a DNA-PK–mediated DDR in the vascular endothelium.

    Table S1. Primer list.

    Data file S1. Sequences for cloning.

    Data file S2. Raw data from figures.

    Movie S1. Transcytosis assay of control gapmeR (25 nM)–transfected HCAECs.

    Movie S2. Transcytosis assay of SNHG12 gapmeR (25 nM)–transfected HCAECs.

    Movie S3. Transcytosis assay of control lentivirus–transduced HCAECs.

    Movie S4. Transcytosis assay of SNHG12 lentivirus–transduced HCAECs.

    Movie S5. Transcytosis assay of control lentivirus–transduced HCAECs in the presence of ROS.

    Movie S6. Transcytosis assay of SNHG12 lentivirus–transduced HCAECs in the presence of ROS.

    References (5963)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Identification and characterization of lncRNA SNHG12 in mouse and human cells.
    • Fig. S2. SNHG12 does not affect lipid metabolism or inflammation.
    • Fig. S3. Identification of DNA-PK as an SNHG12 interactor.
    • Fig. S4. SNHG12 silencing impairs DDR in ECs and macrophages.
    • Fig. S5. Phenotypic effects of SNHG12 on senescence, EC permeability, and efferocytosis.
    • Fig. S6. SNHG12 has no regulatory role in apoptosis.
    • Fig. S7. Accumulating DNA damage and its effect on mitochondrial stress.
    • Fig. S8. Proposed mechanism of SNHG12 regulation of atherosclerosis through a DNA-PK–mediated DDR in the vascular endothelium.
    • Table S1. Primer list.
    • References (5963)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Sequences for cloning.
    • Data file S2 (Microsoft Excel format). Raw data from figures.
    • Movie S1 (.mov format). Transcytosis assay of control gapmeR (25 nM)–transfected HCAECs.
    • Movie S2 (.mov format). Transcytosis assay of SNHG12 gapmeR (25 nM)–transfected HCAECs.
    • Movie S3 (.mov format). Transcytosis assay of control lentivirus–transduced HCAECs.
    • Movie S4 (.mov format). Transcytosis assay of SNHG12 lentivirus–transduced HCAECs.
    • Movie S5 (.mov format). Transcytosis assay of control lentivirus–transduced HCAECs in the presence of ROS.
    • Movie S6 (.mov format). Transcytosis assay of SNHG12 lentivirus–transduced HCAECs in the presence of ROS.

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