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

Abstract

Long noncoding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in atherosclerosis remains poorly defined. We used RNA sequencing to profile lncRNAs derived specifically from the aortic intima of Ldlr−/− mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 (SNHG12) is highly expressed in the vascular endothelium and decreases during lesion progression. SNHG12 knockdown accelerated atherosclerotic lesion formation by 2.4-fold in Ldlr−/− mice by increased DNA damage and senescence in the vascular endothelium, independent of effects on lipid profile or vessel wall inflammation. Conversely, intravenous delivery of SNHG12 protected the tunica intima from DNA damage and atherosclerosis. LncRNA pulldown in combination with liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis showed that SNHG12 interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of SNHG12 reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD+, fully rescued the increases in lesional DNA damage, senescence, and atherosclerosis mediated by SNHG12 knockdown. SNHG12 expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging.

LncRNA VINAS regulates atherosclerosis by modulating NF-κB and MAPK signaling

“…VINAS expression was higher in ECs (isolated from lungs and b.End.3 cell line) compared with other cell types, such as vascular smooth muscle cells (VSMCs) (MOVAS cell line), NIH3T3 fibroblasts, bone marrow-derived macrophages (BMDMs), or the RAW 264.7 macrophage cell line ( Figure 1E), and was broadly expressed in several other organs, with a strong enrichment in the aortic intima compared with the media in the vessel wall ( Figure 1F). Our previous study verified the specificity of aortic intima isolation (15). Therefore, to test whether VINAS lncRNA encodes any protein or peptide, the VINAS sequence was cloned upstream of the p3xFLAG-CMV plasmid, transfected in HEK293 cells, and immunoblotted for FLAG Tag, yielding no detectable peptide or protein ( Figure 1G).…”

“…Arterial inflammation occurs very early in atherogenesis and is associated with impairment of many salutary functions of the healthy endothelium. Accumulating studies point to lncRNAs as regulators of endothelial homeostasis, smooth muscle cell contractility, and macrophage-mediated inflammation in the vessel wall (11,13,(15)(16)(17)(18). This study provides evidence for the first time to our knowledge that the mouse-specific lncRNA VINAS and its human ortholog, DEPDC4, play important roles in vascular inflammation and atherogenesis.…”

“…To explore this expression pattern across species, we analyzed the RNA-Seq data from Yorkshire pigs that were placed for up to 60 weeks on an HCD and developed coronary atherosclerosis. Based on histopathological characterization, the coronary sections were separated into mild, intermediate, and severe groups for progression of atherosclerosis as previously described (15). Similar to VINAS regulation in LDLR -/mice fed an HCD ( Figure 1C), DEPDC4 decreased approximately 60% with disease progression in swine pigs fed an HCD ( Figure 6H).…”

“…Our study expands upon a growing body of literature implicating lncRNAs as pivotal regulators in the development and progression of atherosclerosis. Our group recently identified SNHG12 as an evolutionary conserved lncRNA that plays an important role in atherogenesis (15). SNHG12 mediated the interaction between DNA damage repair proteins DNA-PK and its binding partners Ku70 and Ku80.…”

The Long Non-coding Road to Atherosclerosis

“…Independent of the effects on circulating lipid levels or vessel wall inflammation was the observed accelerated atherosclerosis in high cholesterol diet (HCD)-fed Ldlr −/− mice driven by the knockdown of lncRNA SNHG12 [50]. LncRNA SNHG12 (small nucleolar host gene-12) binds to DNA protein kinase (DNA-PK) in the vascular endothelium, which in turn facilitates binding of DNA-PKcs to Ku70/80 and the ability of DNA damage repair.…”