LncRNA ANRIL acts as a modular scaffold of WDR5 and HDAC3 complexes and promotes alteration of the vascular smooth muscle cell phenotype

Figures

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  Fig. 1. Expression profile of lncRNA ANRIL is enhanced in CAD and induced by ox-LDL. a RNA levels of ANRIL in PBMCs from CAD patients and healthy controls. b, c RNA levels of lncRNA ANRIL in HASMCs treated with ox-LDL, silenced and overexpressed ANRIL measured by qPCR b and FISH stating c. ANRIL in HA **P < 0.01. Antisense probe (AS) of ANRIL was used in FISH, whereas sense probe (S) was included as negative control. CAD, coronary artery disease; HASMC, human aortic smooth muscle cells; FISH, fluorescence in situ hybridization; ox-LDL, oxidized low-density lipoprotein.

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  Fig. 2. LncRNA ANRIL elicits ox-LDL induced phenotypic change in HASMCs. a CCK-8 measured proliferation curves of HASMCs treated by ox-LDL, or transfected with ANRIL overexpressing plasmid and siANRIL. b ROS representative histogram images of flow cytometry findings. c Cellular ROS levels of HASMCs after treated by ox-LDL, or transfected with ANRIL overexpressing plasmid and siANRIL, as detected by flow cytometry. d, e Representative images d and quantification results e of HASMCs with ox-LDL treatment and ANRIL overexpression or silencing, as showed in transwell migration assay. f, g mRNA f and protein g levels of OPN, Collagen type III, Cyclophilin 1, and alph-SMA in HASMCs with ox-LDL inducing and ANRIL overexpressing or silencing. CCK-8, cell counting kit-8; ROS, reactive oxygen species.

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  Fig. 3. LncRNA ANRIL binds and interplays with HDAC3 and WDR5. a, b RNA pull-down assay showing the endogenous proteins HDAC3 and WRD5 interact with biotinylated ANRIL in ox-LDL induced HASMCs, as stained by coomassie blue a and western blots b. c RNA pull-down assay to determinate the binding regions of ANRIL with HDAC3 and WRD5 using truncated ANRIL (Δ1: 1–500, Δ2: 501–1000, Δ3: 1001–1500, Δ4: 1501–2177). d–f Protein immunoprecipitation assay showing ANRIL binds with HDAC3 and WRD5, as pull-downed RNA detected by rapid staining of agarose gels d and qPCR e, f.

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  Fig. 4. The interaction between HDAC3 and WDR5 requires the presence of ANRIL. a–c Protein-protein co-immunoprecipitation assay detecting the interaction between HDAC3 and WDR5 in ox-LDL induced HASMCs a, ox-LDL induced HASMCs with ANRIL silencing b or with RNase treatment c. d Representative fluorescent immunostaining images of HDAC3 and WD5 in ox-LDL induced HASMCs with ANRIL silencing.

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  Fig. 5. ANRIL binds with NOX1 promoter region and actives its expression. a, b mRNA a and protein b levels of NOX1 and NOX4 in HASMCs with ox-LDL inducing and ANRIL overexpressing or silencing. c Dual-luciferase activity assay in which ox-LDL induced HASMCs were transfected with NOX1 gene promoter plasmid and ANRIL overexpressing or silencing plasmids. d Correction analysis of ANRIL and NOX1 expression levels as measured in PBMCs from CAD subjects (n = 45).

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  Fig. 6. NOX1 silencing blocks ox-LOL-induced phenotypic alteration in HASMCs. a CCK-8 measured proliferation curves of HASMCs treated by ox-LDL, or transfected with siANRIL and siNOX1. b Cellular ROS levels of HASMCs after by ox-LDL, or transfected with siANRIL and siNOX1, as detected by flow cytometry. c, d Representative images c and quantification results d of HASMCs with ox-LDL treatment and ANRIL or NOX1 silencing, as showed in transwell migration assay. e, f mRNA e and protein f levels of NOX1 and OPN in HASMCs with ox-LDL inducing and ANRIL or NOX1 silencing.

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  Fig. 7. LncRNA ANRIL coordinates the WDR5/HDAC signals and the associated histone marks in NOX1 promoter region. a NOX1 mRNA levels in HASMCs that transfected with siWD5 (left) and siHDAC3 (right), with or without ANRIL overexpressing plasmid. b, c Chromatin immunoprecipitation (ChIP) detecting WD5/HDAC3 binding with NOX1 promoter DNA in ox-LDL induced HASMCs, as pull-downed DNA detected by rapid staining of agarose gels b and qPCR c. d ChIP detecting histone marks (H3K4me3/H3K9ac) binding with NOX1 promoter DNA in ox-LDL induced HASMCs, as pull-downed DNA detected by rapid staining of agarose gels. e ChIP detecting histone marks (H3K4me3/H3K9ac//H3K27me3) binding with NOX1 promoter DNA in ox-LDL induced HASMCs with silencing of ANRIL, WDR5, or HDAC3. The pull-downed DNA was detected by qPCR. f In vivo interaction between ANRIL with NOX1 promoter was detected in ox-LDL-induced HASMCs by chromatin isolation by RNA purification (ChIRP) assays using DNA antisense oligos(asDNA) specific for ANRIL or a commonly used negative control LacZ. 1, ANRIL asDNA targeting NOX1 promoter region DNA; 2, Negative control LacZ asDNA targeting NOX1 promoter region DNA; 3, ANRIL asDNA targeting GAPDH…