Fig. 3

Transcriptome and dual luciferase assay of Prrx1 and miR-140-3p. A Expression trend analysis of target genes of miR-140-3p in the RM, PC, and CA tissue layers. B GO enrichment analyses of target genes of miR-140-3p. The red arrows represent the most relevant GO terms for antler growth. C Prrx1 expression in the different tissues of sika deer. The values were calculated using public data (accession number CRA002054, publicly accessible at https://ngdc.cncb.ac.cn/gsa). n = 3 biological replicates per group. Data are presented as the mean ± standard error. Two-tailed Student’s t-test was used to compare the differences between two groups. *p < 0.05; **p < 0.01. D Dual-luciferase reporter assay of the binding ability of Prrx1 to the upstream regulatory region of pri-miR-140 identified by ATAC-seq. The red line represents the ATAC peak with Prrx1 binding sites and the nearby regions. The green line represents the ATAC peak with mutant Prrx1 binding sites. The black line represents the putative promotor region. E Dual-luciferase reporter assay of the binding ability of Prrx1 to the upstream regulatory region of pri-miR-140 identified by CUT&Tag-seq. The yellow line represents the CUT&Tag peak with Prrx1 binding sites and the nearby regions. The green line represents the CUT&Tag peak with mutant Prrx1 binding sites. These fragments in D and E were inserted into the front of the luciferase sequence of the pGL3-basic vector in the order indicated in the figure. The relative luciferase activities were measured after cotransfection of these various constructs to 293T cells (n = 3 biological replicates per group). Data are presented as the mean ± standard error. Two-tailed Student’s t-test was used to compare the differences between two groups. *p < 0.05; **p < 0.01. F Dual-luciferase reporter system analysis of the interactions between miR-140-3p and Prrx1. The fragments of putative binding sites [pmirGLO-Prrx1 wild type (WT)] and the corresponding mutated binding sites [pmirGLO-Prrx1 mutated (MUT)] in the 3′ UTRs of Prrx1 gene were selected for plasmid construction. The relative luciferase activities were measured after cotransfection of 293T cells with pmirGLO-Prrx1 constructs that contained putative binding sites or the corresponding mutated binding sites in the 3′-UTRs and either miR-140-3p mimics or negative control for 24 h (n = 3 biological replicates per group). Data are presented as the mean ± standard error. Two-tailed Student’s t-test was used to compare the differences between two groups. *p < 0.05; **p < 0.01