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).