YTHDF2 influences hepatic fibrosis by regulating ferroptosis in hepatic stellate cells by mediating the expression of ACSL4 in an m6A-dependent manner

Abstract

Hepatic fibrosis (HF) is an abnormal reparative response of the liver to chronic injury and is histologically reversible. In recent years, increasing interest has been given to changes in m6A in liver disease. In this study, we explore the role of the m6A-modified reading protein YTHDF2 in HF and its regulatory mechanism. The HF mouse model is generated through CCl4 injection, and the cell model is via TGF-β stimulation. The liver tissues are subjected to hematoxylin-eosin, Masson, and α-SMA immunohistochemical staining. Reactive oxygen species (ROS) and iron levels are examined via relevant kits. Quantitative real-time PCR, immunofluorescence staining, and western blot analysis were conducted to measure the YTHDF2 and ACSL4 levels. RNA immunoprecipitation, methylated RNA immunoprecipitation, RNA pull-down, and polysome fractionation were performed to understand the regulatory mechanism by which YTHDF2 affects ACSL4. The results show that YTHDF2 is highly expressed after HF induction, and the inhibition of YTHDF2 reduces fibrosis as well as ROS and iron levels. In vitro, overexpression of YTHDF2 increases hepatic stellate cell activation, as well as ROS and iron levels, and this effect is blocked by the silencing of ACSL4. YTHDF2 acts as a regulator of ACSL4 expression and is involved in m6A modification. In addition, in vivo experiments indicate that overexpression of ACSL4 reverses the attenuating effect of YTHDF2 interference on HFs. Therefore, YTHDF2 mediates the expression of the ferroptosis marker protein ACSL4 in an m6A-dependent manner, thereby affecting HF.

1. Introduction

Hepatic fibrosis (HF), an injury caused by liver disease, results in the development of scar tissue called fibrosis, resulting in impaired liver function, and has become a serious health problem worldwide [1]. HF is reversible, but when liver fibrosis progresses to the stage of cirrhosis, the prognosis is relatively poor. Thus, revealing the possible molecular events of HF remission is highly important for the development of new therapeutic strategies.

Ferroptosis is triggered when glutathione-dependent antioxidant defenses falter, leading to uncontrolled lipid peroxidation and ultimate cell death [2]. Intracellular iron level affects the development of liver disease and the production of cellular reactive oxygen species (ROS) as well as lipid peroxides [3]. HSCs play a key role in the progression of HF by increasing the production and secretion of the extracellular matrix, whereas ferroptosis contributes to HSC activation and HF progression [4]. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is involved in fatty acid metabolism and synthesis. ACSL4 has been reported to facilitate ferroptosis in tumor cells [5]. In addition, ACSL4 can increase fibrosis and promote ferroptosis in hepatocellular carcinoma (HCC) [6]. Livers lacking ACSL4 exhibit less fibrosis and proliferation, especially in the HCC model of toxic injury [6]. However, further studies are needed to assess whether ACSL4-mediated ferroptosis has a direct effect on HF.

N6-methyladenosine (m6A), considered one of the most common chemical modifications of mRNAs [7], is reversible, dynamically added by the m6A methyltransferase, and cleared away by the demethylase [8]. In addition, m6A exerts multiple biological effects by being directly bound to and recognized by the “reader” proteins of m6A in mRNAs and influences these m6A-modified mRNAs in numerous ways, including their stability, translation, and output [9]. Recent findings indicate that m6A readers play a critical role in the development of liver diseases—for example, the m6A reading protein YTHDF3 mediates PRDX3 translation to alleviate HF [10]. YTHDF2, from the same family, regulates cystathionine-beta-synthase expression in an m6A-dependent manner in gastric cancer, indirectly causing changes in ACSL4 expression [11]. Moreover, YTHDF2 expression is upregulated in HCC and is considered to be a marker of poor prognosis in HCC patients [12]. Therefore, can YTHDF2 directly interfere with ACSL4 expression by regulating the m6A methylation of ACSL4, and does this mode interfere with ferroptosis level and affect HF level?