背景:细胞外基质(ECM)积累升高是纤维化疾病发病机理的主要促成因素。最近的研究表明,N6-甲基腺苷(m6A)RNA修饰在调节RNA稳定性中起关键作用,并有助于各种病理状况的引发。Howbeit,m6A影响ECM沉积的确切机制尚不清楚。
方法:在本研究中,我们使用肥厚性瘢痕(HTSs)作为研究ECM相关疾病的范例。我们专注于ALKBH5介导的m6A去甲基化在HTSs病理进展中的作用,并检查了其与临床分期的相关性。在体内和体外研究了ALKBH5消融对ECM成分的影响。ALKBH5的下游靶标及其潜在机制,使用集成高通量分析确定,RNA结合蛋白免疫沉淀和RNA下拉测定。此外,在纤维化瘢痕模型中评估外源性ALKBH5过表达的治疗潜力.
结果:ALKBH5在来自HTS病变的成纤维细胞中降低,并且与其临床分期呈负相关。重要的是,ALKBH5的消融促进COL3A1、COL1A1和ELN的表达,导致体内和体外ECM的病理沉积和重建。从治疗的角度来看,ALKBH5的外源性过表达可显著抑制纤维化瘢痕模型中胶原的异常沉积。根据集成的高通量分析确定,包括COL3A1、COL1A1和ELN在内的关键ECM组件是ALKBH5的直接下游目标。通过其机制,ALKBH5通过从mRNA中去除m6A抑制COL3A1、COL1A1和ELN的表达,从而以依赖YTHDF1的方式降低它们的稳定性。
结论:我们的研究确定ALKBH5是病理性ECM沉积的内源性抑制因子,有助于开发针对HTS的重新编程的M6A靶向治疗。
BACKGROUND: Elevated extracellular matrix (
ECM) accumulation is a major contributing factor to the pathogenesis of fibrotic diseases. Recent studies have indicated that N6-methyladenosine (m6A) RNA modification plays a pivotal role in modulating RNA stability and contribute to the initiation of various pathological conditions. Howbeit, the precise mechanism by which m6A influences
ECM deposition remains unclear.
METHODS: In this study, we used hypertrophic scars (HTSs) as a paradigm to investigate ECM-related diseases. We focused on the role of ALKBH5-mediated m6A demethylation within the pathological progression of HTSs and examined its correlation with clinical stages. The effects of ALKBH5 ablation on
ECM components were studied both in vivo and in vitro. Downstream targets of ALKBH5, along with their underlying mechanisms, were identified using integrated high-throughput analysis, RNA-binding protein immunoprecipitation and RNA pull-down assays. Furthermore, the therapeutic potential of exogenous ALKBH5 overexpression was evaluated in fibrotic scar models.
RESULTS: ALKBH5 was decreased in fibroblasts derived from HTS lesions and was negatively correlated with their clinical stages. Importantly, ablation of ALKBH5 promoted the expression of COL3A1, COL1A1, and ELN, leading to pathological deposition and reconstruction of the
ECM both in vivo and in vitro. From a therapeutic perspective, the exogenous overexpression of ALKBH5 significantly inhibited abnormal collagen deposition in fibrotic scar models. As determined by integrated high-throughput analysis, key
ECM components including COL3A1, COL1A1, and ELN are direct downstream targets of ALKBH5. By means of its mechanism, ALKBH5 inhibits the expression of COL3A1, COL1A1, and ELN by removing m6A from mRNAs, thereby decreasing their stability in a YTHDF1-dependent manner.
CONCLUSIONS: Our study identified ALKBH5 as an endogenous suppressor of pathological ECM deposition, contributing to the development of a reprogrammed m6A-targeted therapy for HTSs.