N6-Methyladenosine modification (m6A) of circRNA-ZNF638 contributes to the induced activation of SHF stem cells through miR-361-5p/Wnt5a axis in cashmere goats

Abstract

Objective: The objective of this study was to investigate the effects of N6-Methyladenosine modification-circRNA-zinc finger protein 638 (m6A-circRNA-ZNF638) on the induced activation of secondary hair follicle (SHF) stem cells with its potential mechanisms in cashmere goats.

Methods: The m6A modification of ZNF638 was analyzed using methylation immunoprecipitation with real-time quantitative polymerase chain reaction technique in SHF stem cells. The effects of circRNA-ZNF638 on the induced activation of SHF stem cells in m6A dependence were evaluated through the overexpression of circRNA-ZNF638/its m6Adeficient mutants in circRNA-ZNF638 knockdown SHF stem cells. The competitive binding of miR-361-5p to circRNA-ZNF638/Wnt5a 3'- untranslated region was analyzed through Dual-luciferase reporter assay.

Results: The m6A-circRNA-ZNF638 had significantly higher transcription at anagen SHF bulge of cashmere goats compared with that at telogen, as well as it positively regulated the induced activation of SHF-stem cells in cashmere goats. Mechanismly, m6A-circRNA-ZNF638 sponged miR-361-5p to heighten the transcriptional expression of Wnt5a gene in SHFstem cells. We further demonstrated that the internal m6A modification within circRNAZNF638 is required for mediating the miR-361-5p/Wnt5a pathway to regulate the induced activation of SHF stem cells through an introducing of m6A-deficient mutant of circRNAZNF638.

Conclusion: The circRNA-ZNF638 contributes the proper induced activation of SHF-stem cells in cashmere goats in m6A-dependent manner through miR-361-5p/Wnt5a axis.

Keywords: Cashmere Goats; CircRNA-ZNF638; MiR-361-5p; N6-Methyladenosine; SHF Stem Cells; Wnt5a.

Figure 1

The reverse splicing source of circRNA-ZNF638 in cashmere goats with its molecular characteristics. (A) Overall diagram of the host source of circRNA-ZNF638 and its reverse splicing model with the size of 1515-nt. (B) Display of a circRNA-ZNF638 cDNA sequence. The circRNA-ZNF638 cDNA harbors the potential binding sites of five miRNAs indicated by shading with the respective miRNA name where the potential binding region of seed sequence of each miRNA was indicated by underline. Also, it contains four m⁶A modification sites indicated by shading with the same motif of GGACU including m⁶A-374, m⁶A-403, m⁶A-467, and m⁶A-482. circRNA-ZNF638, circRNA-zinc finger protein 638.

Figure 2

The expression analysis of m⁶A-circRNA-ZNF638 in the SHF bugle of cashmere goats and the effects of m⁶A-circRNA-ZNF638 on the induced activation of SHF-stem cells in cashmere goats. (A) Expression pattern of m⁶A-circRNA-ZNF638 in SHF bugle of cashmere goats. (B) Expression pattern of indicator genes on the induced activation of SHF-stem cells in SHF bugle of cashmere goats. (C) Knockdown efficiency analysis of si-circZNF638-1: 5′-ATGTAGTCATTTGAACTTTGTG-3′, si-circZNF638-2: 5′-GAATGTAGTCATTTGAACTTTG -3′, and si-circZNF638-3: 5-TCATTTGAACTTTGTGTTATTC-3′ to m⁶A-circRNA-ZNF638 in SHF-SCs of cashmere goats. (D) Knockdown of m⁶A-circRNA-ZNF638 led to the significant decrease in expression level of the analyzed indictor genes in SHF-stem cells. (E) Enrichment of m6A-modified circRNA-ZNF638 in SHF-stem cells of cashmere goats. The percentage of the input is shown. m⁶A-circRNA-ZNF638, N⁶-Methyladenosine modification-circRNA-zinc finger protein 638; SHF, secondary hair follicle. The asterisk “*” indicates significant difference (p<0.05).

Figure 3

The m⁶A modification of circRNA-ZNF638 is implicated in its contributing to induced activation of SHF-stem cells. (A) Diagram of m⁶A sites within circRNA-ZNF638 sequences of cashmere goat SHFs including m⁶A-374, m⁶A-403, m⁶A-467, and m⁶A-482 (Hui et al [2]). (B) The construction strategies of mutated m⁶A sites for circRNA-ZNF638. circRNA-ZNF638 = wild type of circRNA-ZNF638 (WT), A-G Mutant = m⁶A site mutant of circRNA-ZNF638 (MUT), and NC mutant = negative control mutant. (C) The effects of m⁶A site mutation on circRNA-ZNF638 expression in circRNA-ZNF638 knockdown SHF-stem cells. (D) The effects of m⁶A site mutation on the expression indictor genes in circRNA-ZNF638 knockdown SHF-stem cells. circRNA-ZNF638, circRNA-zinc finger protein 638; SHF, secondary hair follicle. The asterisk (*) stands for a significant difference compared with ‘si-control’ (p<0.05), and the hash mark (#) stands for a significant difference compared with ‘si-circR-1’ or ‘si-circR-2’ (p<0.05). ‘NS’ stands for no significant difference among the different treated groups (p>0.05).

Figure 4

The m⁶A-circRNA-ZNF638 functions as miR-361-5p sponge to enhance the Wnt5a expression SHF-stem cells. (A) The prediction of the binding sites of target miRNAs within m⁶A-circRNA-ZNF638 including miR-103-3p, miR-107-3p, miR-261-5, miR-140-3p, and miR-194. (B) Relative luciferase activities of reporters containing circRNA-ZNF638 in SHF-stem cells 48 h after co-transfection with the indicted different miRNAs. (C) The construction strategies of circRNA-ZNF638 mutant (MUT) for the miR-361-5p binding sites within circRNA-ZNF638. (D) Relative luciferase activities of reporters containing circRNA-ZNF638 mutant (MUT) in SHF-stem cells 48 h after co-transfection with the miR-361-5p or control minics. (E) Expression analysis of miR-361-5p in circRNA-ZNF638 knockdown SHF-stem cells. (F) Expression analysis of Wnt5a mRNA in circRNA-ZNF638 knockdown SHF-stem cells. (G) An overall diagram of goat Wnt5a mRNA along with the prediction of potential binding sites of miR-361-5p on its mRNA 3′-UTR region. The nucleotide positions are indicated according to the goat KLF5 mRNA with accession no. XM_018056510 at NCBI (https://www.ncbi.nlm.nih.gov). (H) Relative luciferase activities of reporters containing Wnt5a 3′-UTR in circRNA-ZNF638 knockdown SHF-stem cells. (I) The construction strategies of Wnt5a mRNA-3′UTR mutant (MUT) for the miR-361-5p binding sites within Wnt5a mRNA-3′UTR. (J) Relative luciferase activities of reporters containing Wnt5a mRNA-3′UTR mutant (MUT) in SHF-stem cells 48 h after co-transfection with the miR-361-5p or control mimics. m⁶A-circRNA-ZNF638, N⁶-Methyladenosine modification-circRNA-zinc finger protein 638; SHF, secondary hair follicle; MUT, mutant. The asterisk “*” indicates significant difference (p<0.05). ‘NS’ stands for no significant difference among the different treated groups (p>0.05).

Figure 5

The m⁶A modification is necessary for circRNA-ZNF638 function through miR-361-5p/Wnt5a pathway that restored the induced activation of SHF stem cells with m⁶A-circRNA-ZNF638-deficient. (A) The effects of m⁶A site mutation on miR-361-5p expression in circRNA-ZNF638 knockdown SHF-stem cells. (B) The effects of m⁶A site mutation on Wnt5a mRNA expression in circRNA-ZNF638 knockdown SHF-stem cells. (C) The miR-361-5p inhibitor led to a significant decrease of its expression in circRNA-ZNF638 knockdown SHF-stem cells. (D) The miR-361-5p inhibitor led to a significant increase of Wnt5a mRNA expression in circRNA-ZNF638 knockdown SHF-stem cells. (E) The miR-361-5p inhibitor led to significantly increased expression of the indictor genes in circRNA-ZNF638 knockdown SHF-stem cells. (F) Overexpression of Wnt5a gene led to significantly increased expression of the indictor genes in circRNA-ZNF638 knockdown SHF-stem cells. m⁶A-circRNA-ZNF638, N⁶-Methyladenosine modification-circRNA-zinc finger protein 638; SHF, secondary hair follicle. The asterisk (*) stands for a significant difference compared with ‘si-control’ (p<0.05), and the hash mark (#) stands for a significant difference compared with ‘si-circR-1’ or ‘si-circR-2’ (p<0.05).

Figure 6

A schematic representation of functional mechanism of m⁶A-circRNA-ZNF638 in contributing to the induced activation of SHF-stem cells in cashmere goats in which m⁶A modification within circRNA-ZNF638 is required for mediating the miR-361-5p/Wnt5a pathway. m⁶A-circRNA-ZNF638, N⁶-Methyladenosine modification-circRNA-zinc finger protein 638; SHF, secondary hair follicle.