Shanghai Jiao Tong University School of Medicine Affiliated Sixth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
PeerJ. 2024 Oct 18;12:e18227. doi: 10.7717/peerj.18227. eCollection 2024.
As one of the most common and abundant internal modifications of eukaryotic mRNA, N-methyladenosine (mA) modifications are closely related to placental development. Ferroptosis is a newly discovered form of programmed cell death. During placental development, placental trophoblasts are susceptible to ferroptosis. However, the interactions of mA and ferroptosis in trophoblast physiology and injury are unclear.
Recurrent miscarriage (RM) was selected as the main gestational disease in this study. Published data (GSE76862) were used to analyze the gene expression profiles in patients with RM. The extent of mA modification in total RNA of villous tissues between patients with RM and healthy controls (HC) was compared. (encoding AlkB homolog 5, RNA demethylase) was selected as the candidate gene for further research. Quantitative real-time reverse transcription PCR, western blotting, and immunohistochemistry (IHC) confirmed the elevated expression of ALKBH5 in the cytotrophoblasts of patients with RM. Then, cell counting kit-8 assays, glutathione disulfide/glutathione quantification, 2',7'-dichlorfluorescein-diacetate staining, and malonaldehyde assays were used to explore the alterations of ferroptosis-related characteristics following RAS-selective lethal (RSL3) stimulation after overexpression of . Thereafter, we re-analyzed the published RNA sequencing data upon knockdown of , combined with published tissue RNA-seq data, and (encoding ferritin light chain) was identified as the ferroptosis-related gene in cytotrophoblasts of patients with RM that is regulated by ALKBH5. Finally, western blotting and IHC confirmed the increased expression of FTL in the cytotrophoblasts from patients with RM.
Total mA levels were decreased in patients with RM. The most significant differentially mA-related gene was , which was increased in patients with RM. cell experiments showed that treatment with RSL3 resulted in increased cell death and upregulated ALKBH5 expression. Overexpression of alleviated RSL3-induced HTR8 cell death and caused decreased levels of intracellular oxidation products. Published transcriptome sequencing revealed that was the major ferroptosis-related gene regulated by ALKBH5 in the villous tissues of patients with RM. Consistent with the expression of ALKBH5, FTL was increased by RSL3-induction and increased in patients with RM.
Elevated ALKBH5 alleviated RSL3-induced cytotrophoblast cell death by promoting the expression of FTL in patients with RM. Our results supported the view that ALKBH5 is an important regulator of the ferroptosis-related etiology of RM and suggested that ALKBH5 could be responsible for epigenetic aberrations in RM pathogenesis.
作为真核 mRNA 中最常见和丰富的内部修饰之一，N6-甲基腺苷（m6A）修饰与胎盘发育密切相关。铁死亡是一种新发现的程序性细胞死亡形式。在胎盘发育过程中，胎盘滋养层细胞容易发生铁死亡。然而，m6A 修饰与滋养层生理学和损伤中的铁死亡之间的相互作用尚不清楚。
本研究选择复发性流产（RM）作为主要妊娠疾病。使用发表的数据（GSE76862）分析 RM 患者的基因表达谱。比较 RM 患者和健康对照者（HC）绒毛组织中总 RNA 中 m6A 修饰的程度。选择（编码 AlkB 同源物 5，RNA 去甲基酶）作为进一步研究的候选基因。定量实时逆转录 PCR、Western blot 和免疫组织化学（IHC）证实 RM 患者的绒毛滋养层中 ALKBH5 的表达升高。然后，使用细胞计数试剂盒-8 测定法、谷胱甘肽二硫化物/谷胱甘肽定量、2'，7'-二氯荧光素二乙酸酯染色和丙二醛测定法，研究过表达后铁死亡相关特征的变化。此后，我们重新分析了敲低后发表的 RNA 测序数据，结合发表的组织 RNA-seq 数据，（编码铁蛋白轻链）被鉴定为受 ALKBH5 调节的 RM 患者绒毛滋养层中的铁死亡相关基因。最后，Western blot 和 IHC 证实了 RM 患者绒毛滋养层中 FTL 的表达增加。
RM 患者的总 m6A 水平降低。差异最显著的 m6A 相关基因是，它在 RM 患者中增加。细胞实验表明，RSL3 处理导致细胞死亡增加和 ALKBH5 表达上调。过表达减轻了 RSL3 诱导的 HTR8 细胞死亡，并导致细胞内氧化产物水平降低。发表的转录组测序显示，在 RM 患者的绒毛组织中，是受 ALKBH5 调节的主要铁死亡相关基因。与 ALKBH5 的表达一致，RSL3 诱导的 FTL 增加，RM 患者中增加。
在 RM 患者中，升高的 ALKBH5 通过促进 FTL 的表达减轻了 RSL3 诱导的滋养层细胞死亡。我们的结果支持 ALKBH5 是 RM 铁死亡相关病因的重要调节剂的观点，并表明 ALKBH5 可能负责 RM 发病机制中的表观遗传异常。
