miR-340-3p-modified bone marrow mesenchymal stem cell-derived exosomes inhibit ferroptosis through METTL3-mediated m6A modification of HMOX1 to promote recovery of injured rat uterus

Abstract

Background Ferroptosis is associated with the pathological progression of hemorrhagic injury and ischemia–reperfusion injury. According to our previous study, exosomes formed through bone marrow mesenchymal stem cells modified with miR-340-3p (MB-exos) can restore damaged endometrium. However, the involvement of ferroptosis in endometrial injury and the effect of MB-exos on ferroptosis remain elusive. Methods The endometrial injury rat model was developed. Exosomes were obtained from the supernatants of bone marrow mesenchymal stromal cells (BMSCs) and miR-340/BMSCs through differential centrifugation. We conducted RNA-seq analysis on endometrial tissues obtained from the PBS and MB-exos groups. Ferroptosis was induced in endometrial stromal cells (ESCs) by treating them with erastin or RSL3, followed by treatment with B-exos or MB-exos. We assessed the endometrial total m6A modification level after injury and subsequent treatment with B-exos or MB-exos by methylation quantification assay. We performed meRIP-qPCR to analyze m6A modification-regulated endogenous mRNAs. Results We reveal that MB-exos facilitate the injured endometrium to recover by suppressing ferroptosis in endometrial stromal cells. The injured endometrium showed significantly upregulated N6-methyladenosine (m6A) modification levels; these levels were attenuated by MB-exos through downregulation of the methylase METTL3. Intriguingly, METTL3 downregulation appears to repress ferroptosis by stabilizing HMOX1 mRNA, thereby potentially elucidating the mechanism through which MB-exos inhibit ferroptosis in ESCs. We identified YTHDF2 as a critical m6A reader protein that contributes to HMOX1 mRNA degradation. YTHDF2 facilitates HMOX1 mRNA degradation by identifying the m6A binding site in the 3′-untranslated regions of HMOX1. In a rat model, treatment with MB-exos ameliorated endometrial injury-induced fibrosis by inhibiting ferroptosis in ESCs. Moreover, METTL3 short hairpin RNA-mediated inhibition of m6A modification enhanced the inhibitory effect of MB-exos on ferroptosis in endometrial injury. Conclusions Thus, these observations provide new insights regarding the molecular mechanisms responsible for endometrial recovery promotion by MB-exos and highlight m6A modification-dependent ferroptosis inhibition as a prospective therapeutic target to attenuate endometrial injury.

1. Introduction

Intrauterine adhesions (IUAs) refer to an iatrogenic condition characterized by the deterioration of functional endometrium in multiple regions, leading to deterioration of the uterine cavity. This condition develops from aggressive or repeated curettages and/or endometritis, resulting in various complications, including amenorrhea, recurrent pregnancy loss, abnormal placentation, infertility, and psychological distress [1]. IUAs incidence in women with infertility ranges from 2 to 22% [2, 3]. Currently, there is no competent method to prevent IUAs reformation. Surgical treatment with lysis of adhesions through hysteroscopy is the primary therapy for IUAs. However, this approach has shown inconsistent results in alleviating symptoms and improving pregnancy rates [2, 4]. Therefore, alternative approaches are required for treating IUA-related infertility.

Ferroptosis, a distinct cell death pathway, results from iron-dependent oxidation of phospholipids containing polyunsaturated fatty acyl tails [5]. As shown recently, ferroptosis participates in the pathological processes of hemorrhagic injury [6]. Morphological changes in mitochondria, a characteristic of ferroptosis, are observed in nerve cells surrounding hematomas, which provides strong evidence for ferroptosis occurrence after cerebral hemorrhage [7]. Ferroptosis inhibition using ferrostatin 1 is a promising approach in reducing hemorrhagic secondary injury and iron deposition in models.