Let-7b in macrophages promotes atherosclerosis progression

Abstract Introduction In macrophages, the endonuclease Dicer produces microRNAs, such as miR-10a and Let-7b, which protect from atherosclerosis by reducing lipid accumulation and apoptosis. While this protective effect of Dicer is partially mediated by miR-10a, the role of macrophage Let-7b in atherosclerosis remains unclear. Methods We created Apoe–/– mice expressing mEGFP in myeloid cells and dTomatored in all other cells with (Apoe–/–ROSAmT/mG/LysM-Cre/Mirlet7bflox/flox, M-mTmG/Let7b–/–) or without a myeloid cell-specific knockout of the Mirlet7b gene (Apoe–/–ROSAmT/mG/LysM-Cre/Mirlet7bWT/WT, M-mTmG/Let7b+/+). Additionally, M-Let7b–/– mice and M-Let7b+/+ mice expressing a GFP-tagged Ago2 in macrophages were generated. Mice were fed a Western-type diet (WD) for 12 or 24 weeks. Cholesterol crystals and macrophages were quantified in fluorescence and polarized light images obtained using the THUNDER imaging system. In situ PCR was performed to detect let-7b expression. Following 12 weeks of diet, GFP-Ago2 immunoprecipitation (IP) was performed on arterial and spleen tissues. Transcript abundance in the GFP-Ago2-IP samples was measured using Prime RNA sequencing. Dual luciferase reporter assays were conducted with HEK293 cells. Let-7b binding sites in the 3’-UTR of target genes were predicted by RNAhybrid. Results We observed that let-7b expression was reduced in plaque macrophages of M-mTmG/Let7b–/– mice compared with control mice. The reduction in aortic mEGFP+ macrophage area after 12 weeks of WD was statistically significant, whereas the crystal area did not reach significance, both measures decreased in M-mTmG/Let7b–/– mice compared to M-mTmG/Let7b+/+ mice (n=6-9) after 24 weeks of WD. In the macrophage Ago2 IP, the abundance of eight out of 739 transcripts was significantly reduced in M-tAgo2/Let7b–/– mice (n=4) compared with M-tAgo2/Let7b+/+ mice (n=3), including microtubule-associated monooxygenase, calponin, and LIM domain-containing 2 (Mical2) and sortilin-related VPS10 domain-containing receptor 2 (Sorcs2). Unlike plaque macrophages, no let-7b-specific targets were detected in spleen macrophages. We predicted three let-7b binding sites in the Mical2 3’-UTR, with one site mediating a reduction in luciferase activity after treatment with let-7b mimic in cells transfected with the Mical2 construct. Conclusion Let-7b expression in macrophages promotes the progression of atherosclerosis by increasing macrophage accumulation. This effect results from let-7b targeting Mical2 and Sorcs2 specifically in plaque macrophages. Therefore, blocking let-7b's targeting in plaque macrophages is a promising strategy to limit atherosclerosis progression.