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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

N6 -methyladenosine (m6 A)-circHECA from secondary hair follicle of cashmere goats: identification, regulatory network and expression regulated potentially by methylation of its host gene promoter

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Objective: The objective of this study was to identify the <i>N</i><sup>6</sup>-methyladenosine (m<sup>6</sup>A)- 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 m<sup>6</sup>A-circHECA and promoter methylation of its host gene (<i>HECA</i>).Methods: The validation of circHECA m<sup>6</sup>A 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 m<sup>6</sup>AcircHECA were performed using SHF stem cells of cashmere goats with RT-qPCR analysis. Based on in-silico analysis, the regulatory networks of m<sup>6</sup>A-circHECA were generated with related signal pathway enrichment. The methylation level of promoter region of m<sup>6</sup>A-circHECA host gene (<i>HECA</i>) was assessed by the bisulfite sequencing PCR (BSPPCR) technique.Results: The m<sup>6</sup>A-circHECA was confirmed to contain four m<sup>6</sup>A modification sites including m<sup>6</sup>A-213, m<sup>6</sup>A-297, m<sup>6</sup>A-780, and m<sup>6</sup>A-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 m<sup>6</sup>A-circHECA of cashmere goats and its potential target molecules: miRNAs, mRNAs, and proteins. The regulatory network and pathway enrichment indicated that m<sup>6</sup>A-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 <i>HECA</i> gene in SHFs of cashmere goats might cause the lower expression of m<sup>6</sup>A-circHECA.Conclusion: The m<sup>6</sup>A-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 m<sup>6</sup>A-circHECA host gene (<i>HECA</i>) most likely was implicated in its expression inhibition in SHFs of cashmere goats.

Abstract

Objective: The objective of this study was to identify the N6 -methyladenosine (m6 A)- 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 m6 A-circHECA and promoter methylation of its host gene (HECA). Methods: The validation of circHECA m6 A 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 m6 AcircHECA were performed using SHF stem cells of cashmere goats with RT-qPCR analysis. Based on in-silico analysis, the regulatory networks of m6 A-circHECA were generated with related signal pathway enrichment. The methylation level of promoter region of m6 A-circHECA host gene (HECA) was assessed by the bisulfite sequencing PCR (BSPPCR) technique. Results: The m6 A-circHECA was confirmed to contain four m6 A modification sites including m6 A-213, m6 A-297, m6 A-780, and m6 A-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 m6 A-circHECA of cashmere goats and its potential target molecules: miRNAs, mRNAs, and proteins. The regulatory network and pathway enrichment indicated that m6 A-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 m6 A-circHECA. Conclusion: The m6 A-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 m6 A-circHECA host gene (HECA) most likely was implicated in its expression inhibition in SHFs of cashmere goats.

참고문헌 (48)

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