Uncovering hidden macrophage small non-coding RNAs associated with atherosclerosis development by PANDORA-seq [bulkRNA-seq]

ABSTRACT: Aim: Small non-coding RNAs (sncRNAs) such as microRNAs (miRNAs) have important functions in the regulation of numerous biological processes. High-throughput RNA sequencing (RNA-seq) method has advanced sncRNA discovery, but RNA modifications have been known to interfere with the cDNA library construction process, leading to the biased sequencing results. To overcome this obstacle, we previously developed an advanced PANDORA-seq method to overcome RNA modification-elicited sequence interferences. PANDORA-seq is able to detect the highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that are otherwise undetectable by traditional RNA-seq protocol. Macrophages are the major immune cells involved in the initiation and progression of atherosclerosis. This study aims to identify novel macrophage sncRNAs associated with atherosclerosis development by using PANDORA-seq. Methods: To identify atherosclerosis-associated macrophage sncRNAs, LDLR-/- mice were fed a low-cholesterol diet (LCD; 0.02% cholesterol) or high-cholesterol diet (HCD; 0.5% cholesterol) for 9 weeks. Total RNAs were isolated from peritoneal macrophages and subjected to both traditional RNA-seq and PANDORA-seq analyses. Results: By overcoming RNA modification-elicited limitations, PANDORA-seq revealed a rsRNA- and tsRNA-enriched sncRNA landscape in the peritoneal macrophages of LDLR-/- mice which was strikingly different from that detected by traditional RNA-seq. Traditional RNA-seq still identified miRNAs as the predominant sncRNA species in macrophages. Strikingly, PANDORA-seq results demonstrated that rsRNAs and tsRNAs were the major macrophage sncRNA types and miRNAs only accounted for ~ 0.2% of total detected sncRNAs. In addition, PANDORA-seq also detected over 286 differentially expressed macrophage sncRNAs induced by HCD feeding. Intriguingly, several HCD-stimulated rsRNAs/tsRNAs promoted pro-atherogenic gene expression in macrophages in vitro, indicating potentially important roles of those “hidden” sncRNAs in atherogenesis. Conclusion: Our findings revealed a comprehensive macrophage sncRNA landscape by uncovering the highly modified rsRNAs/tsRNAs. These abundant rsRNAs/tsRNAs may have important functions in atherosclerosis and warrant future investigations.

ORGANISM(S): Mus musculus

PROVIDER: GSE330468 | GEO | 2026/09/01

REPOSITORIES: GEO