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Differentially regulated miRNAs following murine femoral artery injury

ABSTRACT: We performed wire-induced injuries in 12 week old C57BL/6J mice. Dilation of the femoral artery was performed by inserting a straight spring wire (0.38 mm diameter) for 10 mm towards the iliac artery, as described previously (Sedding D, Daniel JM, Muhl L, Hersemeyer K, Brunsch H, Kemkes-Matthes B, Braun-Dullaeus RC, Tillmanns H, Weimer T, Preissner KT, Kanse SM. The g534e polymorphism of the gene encoding the factor vii-activating protease is associated with cardiovascular risk due to increased neointima formation. The Journal of experimental medicine. 2006;203:2801-2807). Mice were sacrificed and perfused with phosphate buffered saline via the left ventricle. Femoral arteries were excised at 10 and 21 days after injury, snap-frozen and miRNA was isolated by phenol-chloroform extraction following the purelink

ORGANISM(S): Mus musculus

PROVIDER: GSE111701 | GEO | 2019/03/05

REPOSITORIES: GEO

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Project description:Rationale: Neointima formation is a common pathological feature of atherosclerosis and restenosis after angioplasty and involves the proliferation and migration of vascular smooth muscle cells (VSMCs). N6-methyladenosine (m6A), the most prevalent mRNA internal modification and being proposed to be primarily produced by RNA methyltransferase METTL3, plays a vital role in post-transcriptional regulations. Nevertheless, the role of RNA m6A modification in VSMCs and neointima formation remains disputable and undetermined. Objective: To determine the role of METTL3 and its produced RNA modification m6A in VSMCs and neointima formation after vascular injury. Methods and Results: We examined the expression of m6A writers and erasers in the carotid artery collected from human carotid endarterectomy (CEA) as well as in that of mice and unanimously found that METTL3 expression is increased significantly after vascular injury. Then, VSMC-confined METLL3 knockout mice (Myh11CreERT2 METTL3flox/flox) were generated, and carotid artery injury was induced. METTL3 knockout markedly attenuated artery neointima formation induced by wire injury. Moreover, we discovered that METTL3 deficiency repressed both ex vivo and in vivo proliferation of VSMCs. Through a joint analysis of the data from bulk RNA and m6A sequencing, serum- and glucocorticoid-inducible kinase 1 (SGK1) was identified due to its well-documented role in promoting VSMC proliferation and migration. Mechanistically, METTL3-mediated SGK1 mRNA methylation (A146 and A210) was proposed to facilitate SGK1 transcription by recruiting the m6A reader YTHDC1, as shown by well-designed experiments involving methylation site mapping, m6A RNA immunoprecipitation (m6A-RIP), chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) and reporter gene analysis. As anticipated, VSMC proliferation and intimal hyperplasia that had already been mitigated by METTL3 ablation were both restored by SGK1 overexpression. Conclusions: Our findings suggest that METTL3 promotes SGK1 expression via mRNA methylation-mediated facilitation of its own transcription, thus predisposing VSMCs to a proliferative state and contributing to neointima formation after vascular injury, underscoring the essential role METTL3 plays in vascular remodeling.

Project description:IRF9 is ubiquitously expressed and mediates the effects of IFNs, previous study showed that IRF9 played an important role in immunity and cell fate decision. Our recent study revealed that IRF9 involved in cardiac hypertrophy, hepatic steatosis and insulin resistance. However, the function of IRF9 in VSMC and neointima formation was largely unknown. We found that IRF9 expression was significantly increased in the VSMCs of mouse carotid artery. More importantly, we generated SMC-specific IRF9 overexpression transgenic mice (IRF9 TG) and found that IRF9 TG significantly increased VSMC proliferation, migration and neointima formation compared with NTG mice in response to injury. To evaluate the underlying mechanism by which IRF9 promotes VSMC proliferation and migration after vascular injury, IRF9 TG and NTG mice were subjected to wire-injury and the carotid arteries were collected at 14 days post-injury. We combined 3-5 vessels for one sample, and 3 samples for each phenotype. Subsequently, a total of 400ng RNA was used following Affymetrix instruction and 10 ug of cRNA were hybridized for 16 hr at 45°. GeneChips were scanned using the Scanner 7G and the data was analyzed with Expression Console using Affymetrix default analysis settings and global scaling as normalization method. RMA analysis was employed to evaluate the gene expression. We used microarrays to detect the global gene expression in the carotid arteries of smooth muscle cell specific IRF9 transgenic mice(IRF9 TG) compared with non transgenic control mice (NTG) at 14 days post-injury and identified distinct classes of altered genes. non-transgenic controls mice (NTG) and smooth muscle specific IRF9 transgenic mice (IRF9 TG) were subjected to wire-injury and the carotid ateries were collected at 14 days post-injury. We combine 3-5 vessels in one tube and for a single Affymetrix microarray. Total RNA was extracted and a total of 400ng RNA was used following Affymetrix instruction. 3 biological samples for each genotype.