miR-133a-3p and miR-145-5p co-promote goat hair follicle stem cell differentiation by regulating NANOG and SOX9 expression

Objective Hair follicle stem cells (HFSCs) differentiation is a critical physiological progress in skin hair follicle (HF) formation. Goat HFSCs differentiation is one of the essential processes of superior-quality brush hair (SQBH) synthesis. However, knowledge regarding the functions and roles of miR-133a-3p and miR-145-5p in differentiated goat HFSCs is limited. Methods To examine the significance of chi-miR-133a-3p and chi-miR-145-5p in differentiated HFSCs, overexpression and knockdown experiments of miR-133a-3p and miR-145-5p (Mimics and Inhibitors) separately or combined were performed. NANOG, SOX9, and stem cell differentiated markers (β-catenin, C-myc, Keratin 6 [KRT6]) expression levels were detected and analyzed by using real-time quantitative polymerase chain reaction, western blotting, and immunofluorescence assays in differentiated goat HFSCs. Results miR-133a-3p and miR-145-5p inhibit NANOG (a gene recognized in keeping and maintaining the totipotency of embryonic stem cells) expression and promote SOX9 (an important stem cell transcription factor) expression in differentiated stem cells. Functional studies showed that miR-133a-3p and miR-145-5p individually or together overexpression can facilitate goat HFSCs differentiation, whereas suppressing miR-133a-3p and miR-145-5p or both inhibiting can inhibit goat HFSCs differentiation. Conclusion These findings could more completely explain the modulatory function of miR-133a-3p and miR-145-5p in goat HFSCs growth, which also provide more understandings for further investigating goat hair follicle development.


INTRODUCTION
Animals' hair growth is tightly associated with skin hair follicle (HF) formation, and the differentiation of cells in skin hair follicles is critical for hair follicle development, especially the differentiation of hair follicle stem cells [1][2][3].Hair follicle stem cells (HFSCs) are located within the outer root sheaths of hair follicles (HF), which are capable of selfrenewal, maintaining undifferentiated ability, and have proliferative capacity in vivo and in vitro.HFSCs are particularly physiological value in medicine and biology owing to their self-renewal and differentiation activities [4].NANOG, a unique homeobox transcription factor, is one of the key downstream effectors in Wnt and BMP signals [5].NANOG, is also a novel master gene of embryonic stem cell (ESC) pluripotency, which plays vital roles in controlling the pluripotent inner cell mass during embryonic development, sustaining the pluripotent epiblast, and while suppressing primitive endoderm differentiation [6].Besides, NANOG is ubiquitously found in the vigorous divided cells, and as the cell differentiation degree deepens, the expression level of NANOG gradually decreases until it cannot be detected in fully differentiated cells [7,8].SOX9, an important transcription factor, regulates multiple cell type formation, the development of hair follicle (HF), inner ear, testis, heart, kidney, and central nervous system.SOX9 is also a direct modulator of Activin/transforming growth factor beta (TGFβ) signaling related genes [9,10].However, the regulation effect of NANOG and SOX9 in goat HFSCs remains unclear.
Hair follicle development and hair formation are associated with important economic value for commercial applications, such as, Cashmere.Yangtze River Delta white goats' (YRDWG) HFSCs are harvested from newborn male's goat neck skin [11].Our previous research revealed that i) goat HFSCs differentiation is indispensable for superior-quality brush hair (SQBH) formation, this hair is a popular product for its vivid white color, magnificent luster and fine elastic properties, which only can be harvested from YRDWG and is the finest raw material for making Chinese calligraphy brushes [12,13].ii) β-catenin, C-myc, KRT6 can function as the positive regulators of goat HFSCs differentiation, and they were high expression in differentiated stem cells [14].β-Catenin participates in the Wnt/β-catenin axis to activate the downstream effector C-myc and Bmp4 levels, and further inducing HFSCs differentiation.KRT6, a member protein in keratins family, is an important HFSCs differentiation marker [15,16].These studies revealed that β-catenin, C-myc, KRT6 are essential markers and regulatory factors which can influence goat HFSCs differentiation.

Animal care
The animals, materials and experimental procedures used in this study were approved by the Institutional Animal Care and Use Committee, Yangzhou University, Yangzhou, China (Approval ID: SYXK [Su] 2021-0026, number: 202301020).

Cell culture
HFSCs were extracted and cultured from the newborn Yangtze River Delta white goats (YEDWG) neck skin as reported in our prior investigation [11,23], and briefly described below: i) Skin tissues were washed with 0.9% normal saline followed by 75% ethanol with 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) three times.ii) Tissues were then rinsed with phosphate buffered saline (PBS) (Solarbio, Beijing, China) three times and cut into small pieces (approximately 1 mm 3 ).iii) Digestion was performed with 0.25% Trypsin-ethylenediaminetetraacetic acid disodium salt (EDTA) (Gibco, New York, USA) at 37°C for 1.5 h.iv) After digestion, HFs were picked and harvested by means of a stereomicroscope (Leica, Wetzlar, Germany).v) The harvested hair follicles were digested with 0.25% Trypsin-EDTA again at 37°C for 30 min.vi) The digested follicles were placed in Dulbecco's modified eagle medium (DMEM)-F12 supplemented with 20% fetal bovine serum (FBS) and 2% penicillin-streptomycin and were ground in a homogenizer.vii) Finally, the mixed medium was filtered through a 200-mesh cell strainer (Corning, New York, USA) and cultured in 60 mm culture plates at 37°C.Based on this description, we grew HFSCs in 6-well plates (Corning, USA) in 2 mL growth medium consisting DMEM-F12 (Gibco, USA), 20% FBS (Gibco, USA) and 2% penicillinstreptomycin (Invitrogen, USA) with incubation at 37°C in 5% CO 2 .All research protocols received ethical approval from the Yangzhou University.

Cell incorporation
According to our previous study, a β-catenin gene overexpression vector was utilized to stimulate HFSCs differentiation [14].Oligos incorporation was initiated once the stably overexpression β-catenin stem cells reached ~70% to 80% confluency.The miR-133a-3p and miR-145-5p oligos were independently incorporated into the stem cells.Subsequently, the HFSCs were grown, and differentiation was stimulated for up to 5 days.All HFSCs cultures were done in triplicate or higher.
Following Hoechst-33342 (Beyotime, China) nuclear staining, and three times of PBS rinse in the dark, prior to fluorescent images capture via a Leica fluorescence microscope (Leica, Germany).IF quantification employed the ImageJ

chi-miR-133a-3p and chi-miR-145-5p suppress goat HFSCs pluripotency
To examine the significance of chi-miR-133a-3p and chi-miR-145-5p in differentiated HFSCs, overexpression and knockdown experiments of miR-133a-3p and miR-145-5p separately or combined were performed.The efficacy of miR-133a-3p and miR-145-5p oligos was primarily performed in the stem cells.miR-133a-3p and miR-145-5p expression was significantly increased or inhibited by transfection with Mc and inhibitors for 5 days (Supplementary Figure S1A  and 1B).Then, we collected RNA and protein from differentiated stem cells for RT-q-PCR and WB analyses to evaluate the content of the crucial stem cell self-renewing factor and the marker gene of stem cell pluripotency, NANOG.RT-q-PCR and WB analyses revealed that miR-133a-3p and miR-145-5p separately or combined overexpression significantly reduced the mRNA (Figure 1A) and protein (Figure 1B, 1C) expression of NANOG.By contrast, miR-133a-3p and miR-145-5p separately or combined inhibition markedly enhanced the mRNA (Figure 2A) and protein (Figure 2B, 2C) expression of NANOG.These results indicated that miR-133a-3p and miR-145-5p reduce NANOG expression, which might suppress the pluripotency of goat hair follicle stem cells.

DISCUSSION
HFSCs possess more and more potential characteristics in the progression of multilineage differentiation, such as induced to differentiate into smooth muscle cells, keratinocytes, neurons cells and glial cells [29,30].The YRDWG HFSCs, which present important roles in SQBH traits synthesis by interacting with its dermal papilla cells.And meanwhile, goat HF development is determined by the stem cell proliferation and differentiation process.Herein, we demonstrated that i) goat HF development and HFSCs growth are regulated by   some vital proteins and pathway, namely, β-fibrinogen, β-catenin, keratin family protein and Wnt/β-catenin pathway, MAPK pathway [12]; ii) miR-149-5p could target CMTM3 gene and then upregulated AR gene expression, which emerges an essential role during SQBH synthesis by positively facilitating HFSCs proliferation and differentiation [14,23]; iii) Unlike miR-149-5p, circCOL1A1, a circular RNA which sequesters miR-149-5p, plays negative role in HFSCs proliferation and differentiation [24]; DUSP6 gene, also called MKP3 gene, plays critical physiological modulatory functions in MAPK pathway; meanwhile, DUSP6 can be both targeted by miR-133a-3p and miR-145-5p, and miR-133a-3p, and miR-145-5p can suppress HFSCs proliferation via regulation of DUSP6 levels [25,26].Herein, we further investigated the significance of both miRNAs (miR-133a-3p and miR-145-5p) in goat HFSCs differentiation to extensively reveal the modulatory function of miR-133a-3p and miR-145-5p in goat HFSCs growth.miR-133a-3p and miR-145-5p, belong to miR-133 family and miR-145 family, both differentially regulated in produced common-quality brush and SQBH goats, and they exerted low contents in the skin tissues of produced common-quality brush hair goats, and showed higher expression levels in the skin tissues of SQBH goats (Supplementary Figure S2A and  2B).This result suggests that high expression levels of miR-133a-3p and miR-145-5p in skin tissue are beneficial to produce SQBH, which also means miR-133a-3p and miR-145-5p may promote goat skin tissue HFSCs differentiation.Therefore, we speculated that the roles of the two miRNAs roles may be comparable to the miR-149-5p role in goat HFSCs differentiation but play converse roles in regulating the stem cell proliferation.In the other types of cells, miR-133a-3p and miR-145-5p also play different roles.Such as, in myogenic cells, miR-133a could promote myogenic cell proliferation and skeletal muscle development by controlling serum response factor (SRF) and transforming growth factor beta receptor 1 (TGFβR1) expression [31]; miR-145-5p could target SRY-box transcription factor 11 (SOX11) gene and function in tumor-suppressive roles, then to further mediate MYCN proto-oncogene, bHLH transcription factor (MYCN) gene during neuroendocrine differentiation of prostate cancer cells [32].In mouse, miR-24 could limit and inhibit the intrinsic growth ability of HF progenitor by directly targeting phosphoinositide3-kinase (PIK3) gene and reducing the key cycling protein for cell-cycle entry cyclin E1 (CCNE1) expression [33].During HF development, miR-205 is highly enriched in epithelial progenitors and HFSCs, and functions as a HFSCs activator with a vital role in regulating PI3K pathway and HF morphogenesis [34].These known research and facts suggested that same or distinct miRNAs serve varied modulatory function in HF development or the other physiological progresses.
NANOG is a recognized gene in keeping and maintaining the totipotency of ESCs.The major role of NANOG is to prevent the ESC differentiation and keep the cells' totipotency or pluripotency, while the expression level of NANOG need to be downregulated to promote cell differentiation during embryo development [35,36].SOX9 is an important transcriptional and regulatory factor with an extremely conservative HMG domain structure.SOX9 gene determines the generation of HFSCs during the early stage of mouse embryo development.Simultaneously, SOX9 also plays an important role in stem cell differentiation [29,37].Above-mentioned research suggested that NONOG and SOX9 genes are important and play different roles in regulating embryo and HF development.In this research, we demonstrated that miR-133a-3p and miR-145-5p individually or together overexpression in differentiated goat HFSCs decreases NANOG content and increases the expression of SOX9, while individual or together inhibition of miR-133a-3p and miR-145-5p in the differentiated stem cells increases NANOG content, decreases the expression of SOX9.These findings indicated that miR-133a-3p and miR-145-5p might promote goat HFSCs differentiation by reducing NANOG levels and increasing SOX9 expression.In addition, our subsequent stem cell differentiation assays further demonstrated that miR-133a-3p and miR-145-5p individually or together overexpression upregulated β-catenin, C-myc, and KRT6 expressions in goat HFSCs differentiation, whereas miR-133a-3p and miR-145-5p inhibition downregulated the expression level of these stem cell differentiation indicators.This resembles the miR-149-5p function during the differentiation of goat HFSCs [14].
In conclusion, based on our obtained results and these existing studies, we demonstrated that miR-133a-3p and miR-145-5p promote goat HFSCs differentiation by downregulating NANOG expression and upregulating SOX9 and goat HFSCs differentiation bio-markers (β-catenin, C-myc, and KRT6) expression.In addition, these findings could more completely explain the modulatory function of miR-133a-3p and miR-145-5p in goat HFSCs growth, which also provide more understandings or viewpoints for further investigating goat hair follicle development.

Table 1 .
Sequence information for miR-133a-3p and miR-145-5p oligos software.Lastly, integrated optical density (IOD) was computed by dividing the total IOD by the area.

Table 2 .
Primer sequence information for RT-PCR and RT-q-PCR