Fig. 4: Small RNA sequencing revealed that miR-1 is enriched in pTDEs and that its network plays a critical role in cell cycle regulation.

a, b Comparison of the identified miRNAs between pTDEs and mTDEs. c Volcano plot showing differentially expressed miRNAs between pTDEs and mTDEs. The red dots represent pTDE-enriched miRNAs, while the blue dots represent mTDE-enriched miRNAs. The gray dots represent miRNAs with -log10 (p value) and -log2 (fold change) values less than 1.3 and 1, respectively. d Network analysis of miRNAs and their target genes was conducted for the top ten pTDE miRNAs. The size of the colored dots indicates the number of genes regulated by the miRNA. miR-1 has the largest colored dots, which represent more interactions than others. e Comparison of miR-1 expression between CHMp and CHMm cells at the cellular level (left) and in exosomes (right). EVs were treated with RNase for 10 min at 37 °C to degrade contaminating EV-free miRNAs. Threshold cycle (Ct) values were normalized to Uni 6 Spike-in within the same cDNA concentration. f Identification of target genes of miR-1. TargetScan and miRDB were used to screen for miR-1 target genes in the dog database. A Venn diagram indicated that the TargetScan and miRDB datasets shared 293 genes. g, h KEGG and Reactome analyses of the top 100 genes identified via both TargetScan and miRDB. i, j Changes in the expression of potential target genes of miR-1 at the mRNA and protein levels. HACE1, C5orf51, PTPLAD1, GLCCI1, and MMD were decreased when pTDEs were administered. Ct values were used to normalize the levels of target genes to those of A5B, and the starting cDNA concentration was the same for all samples. The protein levels of HACE1 and the HACE1 target gene Rac1 were analyzed via Western blotting. The statistical analysis is presented. The error bars represent the means ± SEMs. Two-way ANOVA was used to compare groups. *P < 0.05, ***P < 0.001.