Department of Infectious Disease, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Department of Pediatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Hepatol Commun. 2023 Aug 3;7(8). doi: 10.1097/HC9.0000000000000199. eCollection 2023 Aug 1.
N6-methyladenosine (m6A), the most prevalent internal RNA modification in eukaryotic cells, is dynamically regulated in response to a wide range of physiological and pathological states. Nonetheless, the involvement of METTL14-induced m6A in liver fibrosis (LF) has yet to be established.
In vitro, HSC cell lines with knock-down and overexpression of METTL14 were constructed, and the effects of METTL14 gene on the phenotypic function of activated HSCs were observed. The proliferation rate was measured by CCK8 and EDU, the cell proliferation cycle was measured by flow detector, the migration rate was measured by Transwell, and the contractility of F-actin was observed after phalloidin staining. The downstream target gene NOVA2 of METTL14 was screened by combined sequencing of MeRIP-seq and RNA-seq, combined with signal analysis. Adeno-associated virus (AAV) was injected into the tail vein in vivo to knock down the expression of METTL14, so as to further observe the role of METTL14 in the progress of LF.
our research showed that the methylase METTL14 content was decreased in hepatic tissue from patients with LF, leading to a lowered degree of m6A modification. Functionally, we discovered that knocking down m6A methyltransferase METTL14 led to increased HSC activation and a substantial worsening of LF. Mechanically, as shown in a multiomics study of HSCs, depleting METTL14 levels decreased m6A deposition onNOVA2 mRNA transcripts, which prompted the activation of YTHDF2 to detect and degrade the decrease of NOVA2 mRNA.
METTL14 functioned as a profibrotic gene by suppressing NOVA2 activity in a mechanism dependent on m6A-YTHDF2. Moreover, knocking down METTL14 exacerbated LF, while NOVA2 prevented its development and partly reversed the damage.
N6-甲基腺嘌呤（m6A）是真核细胞中最普遍的内部 RNA 修饰，可针对广泛的生理和病理状态进行动态调节。然而，METTL14 诱导的 m6A 对肝纤维化（LF）的参与尚未确定。
体外构建 METTL14 敲低和过表达的 HSC 细胞系，并观察 METTL14 基因对激活的 HSCs 表型功能的影响。通过 CCK8 和 EDU 测量细胞增殖率，通过流式细胞仪测量细胞增殖周期，通过 Transwell 测量迁移率，并用鬼笔环肽染色观察 F-肌动蛋白的收缩性。通过 MeRIP-seq 和 RNA-seq 联合测序结合信号分析筛选 METTL14 的下游靶基因 NOVA2。体内通过尾静脉注射腺相关病毒（AAV）敲低 METTL14 的表达，进一步观察 METTL14 在 LF 进展中的作用。
我们的研究表明，LF 患者肝组织中的甲基转移酶 METTL14 含量降低，导致 m6A 修饰程度降低。功能上，我们发现敲低 m6A 甲基转移酶 METTL14 导致 HSC 激活增加，LF 显著恶化。在 HSCs 的多组学研究中，机制表明，耗尽 METTL14 水平会降低 NOVA2 mRNA 转录物上的 m6A 沉积，从而促使 YTHDF2 检测并降解 NOV2 mRNA 的减少。
METTL14 通过抑制 m6A-YTHDF2 依赖的 NOVA2 活性发挥促纤维化基因的作用。此外，敲低 METTL14 加重 LF，而 NOV2 则阻止其发展并部分逆转损伤。
