Inner Mongolian Cashmere Goat Secondary Follicle Development Regulation Research Based on mRNA-miRNA Co-analysis

Abstract

Inner Mongolia cashmere goats, as an important part of animal husbandry production, play an important role in animal fiber industry. In recent years, scientific research has made a lot of explorations on the molecular regulation mechanism of hair follicle cycle growth, but few studies have been reported on the development of cashmere hair in fetal period. This study was based on the completion of 21 skin samples of mRNA and miRNA sequencing in 7 fetal periods (45 days, 55 days,65 days,75 days,95 days,115 days and 135 days) of the Inner Mongolia Cashmere goat. The target genes of miRNA associated with the development of secondary hair follicles in the cashmere goats were selected through the combination analysis of mRNA and miRNA data. Then the overexpression vector was constructed and the interaction between the miRNA and the target gene was identified by Dual-Luciferase Reporter Gene System. The function and interaction relationship of chi-miR-199a-5p and TGF-β2 were verified by RT-qPCR and western blot at the level of the fibroblasts in Inner Mongolia Cashmere goat. It provides a theoretical basis for further study of miRNA and its target genes regulating the occurrence and development of skin hair follicles. As the result shows, the expression trends of 7 genes (BAMBI, SMAD1, LTBP1, PPP2R1B, ID4, BMP8B and PITX2) and 7 miRNA (chi-miR-17-5p, chi-miR-125b-3p, chi-miR-21-5p, chi-miR-143-5p and chi-miR-106b-5p) in the skin samples for the seven stages of the fetus were shown to be consistent with the sequencing results. the results of sequencing are reliable. The correlation coefficient of TGF-β2 and chi-miR-199a-5p in fetal 45d-135d expression is −0.84, showing a strong negative correlation, The target relationship was preliminarily judged. The results of double luciferase vector report showed that chi-miR-199a-5p significantly decreased the expression of luciferase in TGF-β2 3′UTR, It is determined that there is a reciprocal relationship between them at a specific time. We transfected chi-miR199a-5p-FAM mimics into fibroblasts cultured in vitro from Inner Mongolia cashmere goats. After transfection, the cells were harvested to extract total RNA and protein. The mRNA and protein expression levels of TGF-β2 in fibroblasts were detected by RT-qPCR and western blot. It was verified that chi-miR-199a-5p inhibited TGF-β2 expression at both mRNA and protein translation levels in fibroblasts. At the same time, it was again proved that the TGF-β2 gene is a target gene of chi-miR199a-5p.