Rotenone-driven DNA hypermethylation of the miR-6991-3p promoter induces death of mouse brain organoids.

Rotenone has potential chemical toxicity in the nervous system of both insects and mammals, but its deep molecular biological mechanisms have not been clarified. Here, the epigenetic regulatory mechanism underlying the toxicity of rotenone was studied using murine brain organoids (mBOs). Transmission electron microscopy indicated that rotenone destroyed mBOs'mitochondrial structure. RRBS-Seq showed that some promoter regions from the DLK1-DIO3 imprinted microRNA clusters were hypomethylated. But, rotenone stimulated hypermethylation significantly on the promoter DNA of miR-6991-3p. MiR-6991-3p in the rotenone-treated mBOs had the greatest decreased miRNA expression compared with the control. Meanwhile, luciferase report assay indicated that miR-6991-3p induced a decrease in luciferase activity via binding to specific sites on the 3'UTR of DEDD2 gene. To overexpression of miR-6991-3p attenuated mBO proliferated inhibition and cell death, accumulation for lipid peroxidation products significantly by rotenone inducing. Subsequently, results of cell staining and molecular biology experiment revealed that overexpression for miR-6991-3p significantly weakened expression levels of death-related genes (DEDD2, caspase-8, caspase-3, and caspase-1), but significantly elevated expression levels of cell proliferation-related genes (Ki67 and BCL2) in rotenone treated mBOs group. Here, we reveal a novel epigenetic mechanism of rotenone-induced neuronal death, in which rotenone induced promoter DNA hypermethylation of miR-6991-3p in the DLK1-DIO3 imprinted cluster. This caused miR-6991-3p transcriptional activity to be downregulated, which subsequently significantly increased the expression of its target gene, DEDD2, ultimately leading to neural organoid cell death.