Abstract
The Yangtze River Delta white goat is a unique goat species that can produce superior quality brush hair. CKLF-like MARVEL transmembrane domain-containing 3 (CMTM3), which influences the transcriptional activity of androgen receptor (AR), was identified as a candidate gene related to superior-quality brush hair formation. CMTM3 is generally expressed at low levels, but miR-149-5p is highly expressed in the skin tissues of these goats. The mechanism by which CMTM3 regulates the proliferation and apoptosis of goat hair follicle stem cells has not been elucidated. Here, RT-qPCR, western blotting, 5-ethynyl-2′-deoxyuridine (EdU), cell cycle, apoptosis, and dual-luciferase assays were used to investigate the role and regulatory mechanism of CMTM3 and miR-149-5p. Functional studies showed that CMTM3 overexpression inhibited proliferation and induced apoptosis in cultured hair follicle stem cells, whereas silencing CMTM3 markedly facilitated cell proliferation and deterred apoptosis in cultured hair follicle stem cells. Then, using bioinformatic predictions and the aforementioned assays, including dual-luciferase assays, RT-qPCR, and western blotting, we confirmed that miR-149-5p targets CMTM3 and preliminarily investigated the interaction between CMTM3 and AR in goat hair follicle stem cells. Furthermore, miR-149-5p overexpression significantly accelerated the proliferation and attenuated the apoptosis of hair follicle stem cells. Conversely, miR-149-5p inhibition suppressed the proliferation and induced the apoptosis of hair follicle stem cells. These results reveal a miR-149-5p-related regulatory framework for the miR-149-5p/CMTM3/AR axis during superior quality brush hair formation, in which CMTM3 plays a negative role.
Highlights
The Yangtze River Delta white goat, known as the Haimen goat, is the exclusive goat breed in the world that can produce superior-quality brush hair
androgen receptor (AR) expression was higher in superior-quality brush hair goat skin samples than that in normal-quality brush hair goat skin samples (P < 0.05) (Figure 1C) because superior-quality brush hair is only formed in ram goats, and this expression trend is consistent with the RNA-seq results of AR expression in goat hair follicle stem cells after CKLF-like marvel transmembrane domain-containing 3 (CMTM3) interference
We observed that miR-149-5p expression levels were increased in untreated hair follicle stem cells at 24 h, 48 h, 72 h, and 96 h (Figure 1D); the mRNA level of CMTM3 was significantly decreased in untreated hair follicle stem cells at 24 h, 48 h, 72 h, and 96 h (Figure 1E); and the mRNA level of AR was increased over time in untreated hair follicle stem cells, but this change was not significant at 24 h, 48 h, 72 h, and 96 h (Figure 1F)
Summary
The Yangtze River Delta white goat, known as the Haimen goat, is the exclusive goat breed in the world that can produce superior-quality brush hair. This hair is the finest raw material used for making Chinese calligraphy brushes; this breed has been praised for this unique characteristic, which has resulted in it receiving a complimentary name: the brush hair goat (National livestock and Poultry Genetic Resources Committee, 2011). We screened and identified CKLFlike MARVEL transmembrane domain-containing 3 (CMTM3), which modulates the transcriptional activity of androgen receptor (AR), as a putative candidate gene associated with the superiorquality hair trait in Yangtze River Delta white goats (Ji et al, 2018). CMTM3, known as CKLFSF3, is closely related to AR, has a regulatory role in the male reproductive system, and is characterized by its specific leucine zipper domain and “LXXLL” (where L represents leucine and X represents any amino acid) motifs (Zhong et al, 2006)
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