Regulation of Proliferation and Apoptosis of Hair Follicle Stem Cells by miR-145-5p in Yangtze River Delta White Goats

Abstract

Yangtze River Delta white goats are the sole goat breed producing brush hair of high quality. The gene DUSP6 has been extensively studied in tumor cells but rarely in hair follicle stem cells (HFSCs). Per the previous sequencing data, it was determined that DUSP6 expression was up-regulated in superior-quality brush hair tissues, confirming it as a candidate gene associated with this trait. The targeting relationship of miR-145-5p with DUSP6 was determined based on online database prediction and was authenticated using a dual-luciferase gene reporter assay and quantitative reverse-transcription PCR (RT-qPCR). The regulatory effect of miR-145-5p on the growth of HFSCs was determined by targeting DUSP6 with RT-qPCR, 5-ethynyl-2'-deoxyuridine assays, Western blotting, and flow cytometry. The proliferation of HFSCs was inhibited and their apoptosis capacity was enhanced due to the presence of miR-145-5p. Therefore, it was proposed that this may have occurred through a repression effect of DUSP6 on the MAPK signaling pathway. The regulatory network of the HFSCs can be further understood using the theoretical basis established by the findings derived from this study.

Keywords: DUSP6; brush hair; goat; miR-145-5p; proliferation.

Figure 1

Inhibition of stem cell proliferation of goat hair follicles by miR-145-5p. (A) RT-qPCR quantified the expression of marker genes (CDK1, PCNA, CCND2) following miR-145-5p overexpression. (B) Western blotting showed the expression of proliferation marker genes (CDK1, PCNA, CCND2) following transfection with miR-145-5p mimics. (C) Cell cycle detection by flow cytometry following miR-145-5p upregulation. (D,E) The distribution of the three phases. (F) Distribution and rate of EdU-positive cells. No asterisk, p > 0.05; *, p < 0.05.

Figure 2

miR-145-5p knockdown promotes goat HFSC proliferation. (A) RT-qPCR quantified the expression of marker genes (CDK1, PCNA, CCND2) following miR-145-5p downregulation. (B) Western blotting showed the proliferation-associated marker genes’ expression levels (CDK1, PCNA, CCND2) following transfection with miR-145-5p inhibitors. (C) Cell cycle detection by flow cytometry following miR-145-5p knockdown. (D,E) The distribution of the three phases. (F) Distribution and rate of EdU-positive cells. No asterisk, p > 0.05; *, p < 0.05; **, p < 0.01.

Figure 3

miR-145-5p regulates goat HFSC apoptosis. (A) RT-qPCR quantified the expression of marker genes (BAX and BCL2) following miR-145-5p upregulation. (B) Western blotting showed the expression of proliferation marker genes following transfection with miR-145-5p mimics. (C) RT-qPCR was utilized to quantify the marker genes’ expression (BAX and BCL2) following miR-145-5p downregulation. (D) Western blot analysis of the expression of proliferation marker genes following transfection with miR-145-5p inhibitors. (E–H) Distribution and proportion of apoptotic cells in the upgrade and knockdown groups. No asterisk, p > 0.05; *, p < 0.05; **, p < 0.01.

Figure 4

Relationship between DUSP6 and miR-145-5p. (A) Dual luciferase gene reporter assay of DUSP6 wild-type vector and DUSP6 mutant-type vector transfected with miR-145-5p mimics. (B,C) Protein expression of DUSP6 in the upgrade and knockdown groups. No asterisk, p > 0.05; *, p < 0.05.