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

Chengxin Zhang,Shangqing Ge,Zhixiang Guo,Xiaotian Gao,Xiaoyong Wei,Shenglin Ge,Zhuang Liu,Wenhui Gong,Jinguo Xu

doi:10.1038/s41419-020-2645-3

Abstract

Many studies have shown that long-noncoding RNA (lncRNA) is associated with cardiovascular disease, but its molecular mechanism is still unclear. In this study, we explored the role of lncRNA ANRIL in ox-LDL-induced phenotypic transition of human aortic smooth muscle cells (HASMC). The results of quantitative fluorescence PCR showed that the expression of ANRIL in patients with coronary atherosclerotic heart disease (CAD) was significantly higher than that in normal subjects. RNA-FISH detection showed that the ANRIL expression increased in HASMC treated by ox-LDL. Ox-LDL could upregulate the expression of ANRIL and ROS and promote the phenotypic transition of HASMC. After downregulation of ANRIL by siRNA, ROS level decreased and HASMC phenotypic transition alleviated. ANRIL could act as a molecular scaffold to promote the binding of WDR5 and HDAC3 to form WDR5 and HDAC3 complexes, they regulated target genes such as NOX1 expression by histone modification, upregulated ROS level and promote HASMC phenotype transition. Therefore, we found a new epigenetic regulatory mechanism for phenotype transition of VSMC, ANRIL was a treatment target of occlusive vascular diseases.

Highlights

Coronary aortic disease (CAD) represents a series of life-threatening disorders characterized by formation of foam cells and atherosclerotic plaques on aortic wall[1]
Our results demonstrated antisense noncoding RNA in the INK4 locus (ANRIL) serves as a potential scaffold protein interacts with WDR5 and HDAC3 histone modifying complex and affects epigenetic regulatory signals on
ARNIL transcripts was found adjacent to chromosome 9p21 (Chr9p21) and the genetic variation as well as its expression level was directly correlated with severity of atherosclerosis[31,32]

Summary

Result

LncRNA ANRIL is a large noncoding RNA previously reported to exist as a genetic susceptibility locus with inverse correlation to coronary disease, type 2 diabetes, and several types of cancer[19,20,21,22,23]. We asked whether NOX1 is involved in ANRIL promoted phenotype switch in HASMCs. The results showed the enhanced cell proliferation, ROS production and migration activity after ox-LDL treatment were all attenuated significantly by siRNA-mediated repression of NOX1 (Fig. 6a–d). Similar change was observed in expression level of synthetic phenotype marker OPN, together suggesting the critical role of NOX1 to facilitate the phenotype switch of smooth muscle cells of LncRNA regulator ANRIL (Fig. 6e, f). Significant decrease of these binding signals was observed in ANRIL-silenced cells (Fig. 7c, e), suggesting ANRIL coordinates the localization of WDR5/ HDAC3 and regulates the associated epigenetic marks in downstream promoter element. Confirmed by chromatin isolation by RNA purification (ChIRP) assay, whereas no RNA-DNA binding was detected between ANRIL with NOX1 intron DNA, promoter DNA of control gene GAPDH or a control RNA LacZ with NOX1 promoter DNA (Fig. 7f)

Discussion

Findings

Materials and methods