N6-methyladenosine (m6A)-circHECA from secondary hair follicle of cashmere goats: identification, regulatory network and expression regulated potentially by methylation of its host gene promoter

Objective: The objective of this study was to identify the N⁶-methyladenosine (m⁶A)- circHECA molecule in secondary hair follicles (SHFs) of cashmere goats, and generate its potential regulatory network, as well as explore the potential relationship between transcriptional pattern of m⁶A-circHECA and promoter methylation of its host gene (HECA).Methods: The validation of circHECA m⁶A sites was performed using methylation immunoprecipitation (Me-RIP) along with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technique. The nucleus and cytoplasm localizations of m⁶AcircHECA were performed using SHF stem cells of cashmere goats with RT-qPCR analysis. Based on in-silico analysis, the regulatory networks of m⁶A-circHECA were generated with related signal pathway enrichment. The methylation level of promoter region of m⁶A-circHECA host gene (HECA) was assessed by the bisulfite sequencing PCR (BSPPCR) technique.Results: The m⁶A-circHECA was confirmed to contain four m⁶A modification sites including m⁶A-213, m⁶A-297, m⁶A-780, and m⁶A-927, and it was detected mainly in cytoplasm of the SHF stem cells of cashmere goats. The integrated regulatory network analysis showed directly or indirectly complex regulatory relationships between m⁶A-circHECA of cashmere goats and its potential target molecules: miRNAs, mRNAs, and proteins. The regulatory network and pathway enrichment indicated that m⁶A-circHECA might play multiple roles in the SHF physiology process of cashmere goats through directly or indirectly interacting or regulating its potential target molecules. A higher methylation level of promoter region of HECA gene in SHFs of cashmere goats might cause the lower expression of m⁶A-circHECA.Conclusion: The m⁶A-circHECA might play multiple roles in SHF physiology process of cashmere goats through miRNA mediated pathways along with directly or indirectly interaction with its target proteins. The promoter methylation of m⁶A-circHECA host gene (HECA) most likely was implicated in its expression inhibition in SHFs of cashmere goats.