m6A-methylated TAL1 exacerbates lipid accumulation in ethylene bisdithiocarbamate metabolite–induced anorectal malformations in rat fetuses via miR-205/LCOR signaling

Abstract

Background

Ethylene thiourea (ETU), the primary metabolite of the ethylene bisdithiocarbamate (EBDC) class of fungicides, has several harmful health effects. Anorectal malformations (ARMs), which can be induced by ETU in rats, are congenital conditions in humans that often lead to postoperative defecation dysfunction despite advances in surgical methods. Spinal defecation center dysplasia has been observed during intrauterine development. However, the underlying pathological mechanisms remain unclear.

Methods

The potential molecular mechanisms were investigated using bioinformatics, RNA stability, RNA immunoprecipitation (RIP) assay, chromatin immunoprecipitation (ChIP)–quantitative real-time polymerase chain reaction (ChIP-qPCR), and dual-luciferase reporter assays in HEK 293 T cells. Western blotting and qRT-PCR were used to explore the biological functions of the indicators in HEK 293 T cells and samples from ETU-induced ARM fetal rat models. Transamniotic microinjection was performed in fetal rat models with ARM.

Results

Abnormally elevated microRNA (miR)-205 levels were observed in fetal rats with ARMs, and miR-205 inhibited LCOR expression by binding to its 3’-UTR. Subsequently, TAL1 was identified through bioinformatic analysis as an upstream transcriptional regulator of miR-205. TAL1 positively regulates miR-205 transcription by binding directly to a novel AGATAAG motif in the miR-205 promoter. In addition, TAL1 upregulation was demonstrated to be stabilized by IGF2BP1 in an m⁶A-dependent manner. Mechanistically, IGF2BP1 serves as a reader for m⁶A-modified TAL1 through its RRM1–2 domain directly binding with TAL1 and modification at 1549 adenosine within the m⁶A motif “GGACU” of TAL1. Functionally, IGF2BP1-stabilized TAL1 promotes lipid accumulation by activating the miR-205–LCOR axis. Notably, intra-amniotic microinjection of LCOR restored excessive lipogenesis in rat embryos with ARMs.

Conclusions

These findings indicate that the novel IGF2BP1/TAL1/miR-205/LCOR axis leads to lipid accumulation. Additionally, intra-amniotic injection of LCOR helps restore abnormal lipid metabolism in the lumbosacral defecation center of rats with ETU-induced ARMs during embryogenesis.