DNA methylation of microRNA-365-1 induces apoptosis by targeting DAP3 to induce apoptosis production in hair follicle stem cells

Background DNA methylation is a crucial epigenetic alteration implicated in diverse biological processes and illnesses. Nevertheless, the precise involvement of DNA methylation in chemotherapeutic alopecia remains unclear. This study aimed to examine the role and novel processes of DNA methylation in the regulation of chemotherapeutic drug-induced alopecia. Methods A mouse alopecia model induced by cyclophosphamide (CTX) was established for this study. Hematoxylin-eosin staining was employed to assess the structural integrity of hair follicle stem cells. The proliferative efficiency of these cells was evaluated through immunohistochemical staining for the proportion of Ki67 and a mitochondrial membrane potential assay (JC-1). Immunofluorescence and real-time fluorescence quantitative PCR (RT-qPCR) were utilized to determine the expression levels of key hair follicle stem cell markers, namely Lgr5, CD49f, Sox9, CD200, and FZD10. Differential DNA methylation levels between normal and CTX-induced model groups were investigated using simple methylation sequencing and analyzed through bioinformatics tools. The expression levels of miR-365-1, apoptosis markers, and DAP3 were detected through RT-qPCR and Western blotting (WB). In parallel, primary mouse hair follicle stem cells were extracted and utilized as a cell model. The cell model was constructed using 4-hydroperoxycyclophosphamide (4-HC), and the luciferase reporter gene assay was employed to confirm the binding of miR-365-1 to DAP3. To measure the expression of relevant indicators, superoxide dismutase (SOD) and malondialdehyde (MDA) kits were employed. Methylation-specific PCR (MS-PCR) was utilized to identify the level of DNA methylation. The regulatory relationship within hair follicle stem cells was confirmed through siRNA knockdown of miR-365-1 and plasmid overexpression of DAP3. Result In the alopecia areata model induced by cyclophosphamide (CTX), a substantial number of apoptotic cells were observed within the mouse hair follicles on their backs. Immunofluorescence staining indicated a significant reduction in the expression of hair follicle stem cell markers in the CTX model group. Both RT-qPCR and Western blot results demonstrated a noteworthy difference in the expression of DNA methyltransferase. Analysis from simple methylation sequencing unveiled a substantial increase in DNA methylation within the dorsal skin of mice in the CTX group. Subsequent screening identified miR-365-1 as the most differentially expressed, which was predicted and confirmed to bind to the target gene DAP3. In the CTX group, there was a marked reduction in the expression of superoxide dismutase (SOD) and ATP, while malondialdehyde (MDA) levels were significantly elevated. Cellular investigations revealed that 4-HC-induced cell cycle arrest and a decrease in the expression of hair follicle stem cell markers in hair follicle stem cells. Methylation-specific PCR (MS-PCR) indicated hypermethylation modification of miR-365-1 in 4-HC-induced hair follicle stem cells. A luciferase reporter gene experiment confirmed the presence of miR-365-1 binding to the DAP3 promoter region. Overexpression of miR-365-1 led to a dramatic reduction in the expression of apoptotic proteins in hair follicle stem cells. However, this effect was slightly reversed after lentiviral overexpression of DAP3. Conclusion In this study, we explored the occurrence of miR-365-1 DNA methylation in chemotherapeutic alopecia and found that miR-365-1 reduces apoptosis by targeting DAP3 in hair follicle stem cells, revealing the role of DNA methylation of the miR-365-1 promoter in chemotherapeutic alopecia.