Chi-miR-370-3p regulates hair follicle morphogenesis of Inner Mongolian cashmere goats

Abstract

MicroRNAs (miRNAs), a class of 22 nucleotide (nt) noncoding RNAs, negatively regulate mRNA posttranscriptional modification in various biological processes. Morphogenesis of skin hair follicles in cashmere goats is a dynamic process involving many key signaling molecules, but the associated cellular biological mechanisms induced by these key signaling molecules have not been reported. In this study, differential expression, bioinformatics, and Gene Ontology/Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed on miRNA expression profiles of Inner Mongolian cashmere goats at 45, 55, and 65 days during the fetal period, and chi-miR-370-3p was identified and investigated further. Real-time fluorescence quantification (qRT-PCR), dual luciferase reporting, and Western blotting results showed that transforming growth factor beta receptor 2 (TGF-βR2) and fibroblast growth factor receptor 2 (FGFR2) were the target genes of chi-miR-370-3p. Chi-miR-370-3p also regulated the expression of TGF-βR2 and FGFR2 at mRNA and protein levels in epithelial cells and dermal fibroblasts. DNA staining, Cell Counting Kit-8, and fluorescein-labelled Annexin V results showed that chi-miR-370-3p inhibited the proliferation of epithelial cells and fibroblasts but had no effect on apoptosis. Cell scratch test results showed that chi-miR-370-3p promoted the migration of epithelial cells and fibroblasts. Chi-miR-370-3p inhibits the proliferation of epithelial cells and fibroblasts by targeting TGF-βR2 and FGFR2, thereby improving cell migration ability and ultimately regulating the fate of epithelial cells and dermal fibroblasts to develop the placode and dermal condensate, inducing hair follicle morphogenesis.

Keywords: cashmere goat; cell migration; cell proliferation; chi-miR-370-3p; hair follicle.

Figure 1

Screening of miRNAs related to fetal skin hair follicle morphogenesis in Inner Mongolian cashmere goats. (A) miRNAs differentially expressed at 55 and 45 days. (B) miRNAs differentially expressed at 55 and 65 days. (C) Heatmap of miRNAs related to DC and PC initiation. (D) GO functional annotation and enrichment analysis. The top 10 GO terms in each category are shown in the figure. (E) KEGG signaling pathway enrichment analysis.

Figure 2

Verification of chi-miR-370-3p sequencing results and confirmation that it directly targets TGF-βR2 and FGFR2. (A) Validation of chi-miR-370-3p sequencing results. (B) qRT-PCR verification of the targeting of TGF-βR2 and FGFR2 by chi-miR-370-3p. (C) Plasmid map of the dual luciferase reporter system and location of the target fragment inserted into the vector. (D) Verification of the interaction between chi-miR-370-3p and TGF-βR2-3′-UTR by dual luciferase reporter gene assay. (E) Verification of the interaction between chi-miR-370-3p and FGFR2-3′-UTR by dual luciferase reporter gene assay.

Figure 3

Verification of the regulatory effect of chi-mir-370-3p on TGF-βR2 and FGFR2 at epithelial cell and dermal fibroblast levels. (A) Construction of chi-miR-370-3p (lo) and chi-miR-370-3p (hi) dermal fibroblast and epithelial cell lines. (B) Relative expression of chi-miR-370-3p in various cell lines. (C) Relative expression of TGF-βR2 and FGFR2 in various cell lines. (D) Expression of β-actin, TGF-βR2, and FGFR2 proteins in each cell line. (E) Relative abundance of TGF-βR2 and FGFR2 proteins in different epithelial cell lines. (F) Relative abundance of TGF-βR2 and FGFR2 proteins in different dermal fibroblast cell lines.

Figure 4

Effects of chi-miR-370-3p on the proliferation of epithelial cells and dermal fibroblasts. (A) Cell cycle analysis by flow cytometry. (B) Statistical analysis of the cell cycle in each cell line (**P < 0.01). (C) Increment index of each cell line. (D) Cell growth curve for each epithelial cell line. (E) Cell growth curve for each dermal fibroblast cell line.

Figure 5

Effects of chi-miR-370-3p on apoptosis and migration of epithelial cells and dermal fibroblasts. (A) Apoptosis analysis by flow cytometry. (B) Apoptosis rate of each cell line. (C) Effect of serum-free medium on the proliferation of each cell line over 24 h. (D) Results of cell scratch tests. (E) Cell migration rate of each cell line.