Transcriptome-wide analysis of 5-methylcytosine modification and expression profiles in a mouse model of cerebral ischemia

Stroke is one of the leading causes of death and disability, surpassing heart disease and respiratory diseases, and has become the primary cause of disability-adjusted life years lost in patients. Ischemic stroke (IS) accounts for 87% of all stroke cases. Epigenetics refers to the heritable and modifiable processes that regulate gene expression without altering the DNA sequence. This includes DNA methylation or hydroxymethylation, histone modifications, chromatin remodeling, and RNA methylation. 5-methylcytosine (m5C) is one form of RNA methylation that has been reported to be widely present in various RNAs. Studies have shown that m5C modification is involved in several fields, including cancer, cardiovascular diseases, reproductive diseases, and neurological diseases. However, the specific role of m5C methylation in stroke remains unclear. Therefore, We set up the mouse cerebral ischemia model (Middle Cerebral Artery Occlusion, MCAO) to study the epigenetic modifications and potential biological functions of relevant RNAs after stroke. Overall design: First, we build the cerebral ischemia model of the male c57 mice, divided into a sham surgery group (Sham) and a stroke group (MCAO). After model establishment, we validated the model using TTC staining, TUNEL staining, and immunofluorescence staining. We assessed the extent of nerve damage in mice after MCAO using the Longa neurological deficit score. We used methylated RNA immunoprecipitation sequencing (MeRIP-Seq) to identify m5C peaks in the mouse transcriptome (lncRNA, circRNA, mRNA), and simultaneously constructed RNA libraries through RNA-Seq to study the expression profile. We analyzed the differentially methylated lncRNA, circRNA, and mRNA, and studied their potential biological functions through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The final validation was performed using RT-PCR

脑卒中是全球致死与致残的主要病因之一，其致死、致残率已超过心脏病与呼吸系统疾病，亦是导致患者伤残调整生命年损失的首要原因。缺血性脑卒中（Ischemic Stroke, IS）占所有脑卒中病例的87%。表观遗传学（Epigenetics）指的是在不改变DNA序列的前提下，调控基因表达的可遗传、可修饰的生物学过程，涵盖DNA甲基化、羟甲基化、组蛋白修饰、染色质重塑以及RNA甲基化。5-甲基胞嘧啶（5-methylcytosine, m5C）作为RNA甲基化的一种类型，据报道广泛存在于各类RNA中。已有研究表明，m5C修饰参与癌症、心血管疾病、生殖系统疾病及神经系统疾病等多个研究领域，但m5C甲基化在脑卒中中的具体作用仍不明确。因此，本研究构建小鼠脑缺血模型（大脑中动脉阻塞，Middle Cerebral Artery Occlusion, MCAO），以探究脑卒中后相关RNA的表观遗传修饰模式及其潜在生物学功能。整体实验设计如下：首先构建雄性C57小鼠脑缺血模型，将其分为假手术组（Sham）与脑卒中模型组（MCAO）。模型构建完成后，通过TTC染色、TUNEL染色与免疫荧光染色对模型进行验证。采用Longa神经功能缺损评分评估MCAO模型小鼠的神经损伤程度。利用甲基化RNA免疫共沉淀测序（Methylated RNA Immunoprecipitation Sequencing, MeRIP-Seq）鉴定小鼠转录组（长链非编码RNA（long non-coding RNA, lncRNA）、环状RNA（circular RNA, circRNA）、信使RNA（messenger RNA, mRNA））中的m5C修饰峰，同时通过RNA测序（RNA-Seq）构建RNA文库以分析其表达谱。对差异甲基化的长链非编码RNA、环状RNA及信使RNA进行分析，并通过基因本体论（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析探究其潜在生物学功能。最终采用逆转录聚合酶链式反应（RT-PCR）进行验证。