Investigating epigenetic changes in slovene male suicides: insights from chip-seq and micro rna analysis

To understand suicide and suicidal behavior, we investigated epigenetic changes in the post-mortem tissue of Slovene male suicides. Epigenetics refers to changes in DNA methylation, histone tail modifications, and non-coding RNAs. The first two alter the accessibility of genes to the regulatory elements and the last regulates transcribed RNAs. Unlike genetic changes, epigenetic changes are dynamic and respond to environmental factors. Lately, many studies showed that epigenetic changes contribute to the development of psychopathology and suicidal behavior. For the purpose of studying epigenetic changes of the suicides, we performed chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), and micro RNA (miRNA) analysis. Firstly, with the ChIP-seq experiment, we investigated the acetylation of the lysine 14 on histone 3 (H3K14ac) modification in the hippocampus of a group of suicides and controls. DNA precipitated with histone proteins was sequenced and analyzed. Furthermore, genes associated with differential H3K14ac enrichment in the upstream position of the gene and with the lowest q-value will be analyzed with qPCR. Secondly, we investigated miRNAs from extracellular vesicles (EVs) of the cerebrospinal fluid (CSF) from the suicide and control group. EVs were isolated with ultracentrifugation, and characterized with nanoparticle tracking analysis and Western blot. Then, qPCR was used for investigating the presence of selected miRNAs in the EVs. The study of H3K14ac showed an overall decrease in acetylation of lysine 14 in the hippocampus of suicides. From 293046 peaks, 26894 peaks reached statistical significance (q ≤ 10-3). Peaks with statistical significance were associated with the closest genes. Among genes with H3K14ac difference (q < 0.05) were genes related to suicide; SLCA6A4, MAOA, DDC, COMT, FKBP5, NR3C1, NTRK2, AKT1, APOE, SAT1. A study of suicide or depression-related miRNAs showed that from 20 miRNAs, 9 were present in EVs of the CSF. We found that miR-19a-3p and miR-4516 reached statistical significance (p < 0.05). MiR-4516 and miR-19a-3p have been previously studied in suicide, and target SLC6A4 and TNF-α expression, correspondingly. We showed that H3K14ac is decreased through genome in the group of suicides, which is in accordance with our expectations. Namely, studies of histone tail modifications in depression, which is one of the common comorbidities of suicide, showed lower levels of H3K14ac in depressed subjects compared to controls. Since H3K14ac is associated with opened chromatin, we expect that genes with lowered H3K14ac in a position upstream of the gene have also lowered gene expression. Therefore, on selected genes with the lowest q-value, we will perform qPCR. EVs transfer information (proteins, miRNAs…) from one cell to another. The content of the EVs and their target cell is thought to be selected. We showed that not all miRNAs from the brain were detected in EVs of the CSF. Furthermore, EVs cargo should be investigated in more detail, since EVs can also cross the blood-brain barrier.