MicroRNA-148b promotes proliferation of hair follicle cells by targeting NFAT5

MicroRNAs (miRNAs), small non-coding RNAs, are involved in many aspects of biological processes. Previous studies have indicated that miRNAs are important for hair follicle development and growth. In our study, we found by qRT-PCR that miR-148b was significantly upregulated in sheep wool follicle bulbs in anagen phase compared with the telogen phase of the hair follicle cycle. Overexpression of miR-148b promoted proliferation of both HHDPC and HHGMC. By using the TOPFlash system we demonstrated that miR-148b could activate Wnt/β-catenin pathway and b-catenin, cycD, c-jun and PPARD were consistently upregulated accordingly. Furthermore, transcript factor nuclear factor of activated T cells type 5 (NFAT5) and Wnt10b were predicted to be the target of miR-148b and this was substantiated using a Dual-Luciferase reporter system. Subsequently NFAT5 was further identified as the target of miR-148b using western blotting. These results were considered to indicate that miR-148b could activate the Wnt/β-catenin signal pathway by targeting NFAT5 to promote the proliferation of human hair follicle cells.


Introduction
Hairs, unique to mammals, are organs found in the skin and have various functions [1,2] . Unlike other organs during the postnatal life, hair follicles undergo a cyclic period including growth (anagen), regression (catagen) and quiescence (telogen), in which a large number of molecular signals participate. Anagen is characterized by cell proliferation in the proximal hair follicle epithelium, which is a unique process of programmed organ regeneration [2,3] . The length of the hair is determined by the duration of anagen, which is dependent upon the incessant proliferation and differentiation of the matrix cells at the follicle base known as transit-amplifying cells [4] . Catagen, a highly controlled process of coordinated cell differentiation and apoptosis, is followed by telogen in which the follicles lie dormant and stay in the upper dermis [5] . The continuous postnatal follicle growth cycling has drawn public attention, and subsequently the molecular mechanisms by which the follicles regenerate and regress have been elucidated. Previous studies have shown that many signal pathways are involved in the process of the hair growth cycling, including Wnt/β-catenin, BMP, FGF and Shh signaling [6][7][8][9][10][11][12] . Wnt signaling is involved in many aspects of embryonic development as well as maintaining homeostatic self-regeneration in numerous adult tissues [13] . β-catenin, a key signaling molecule in the Wnt/ β-catenin pathway, is stabilized in the cytoplasm and then enters cell nuclei to mediate the transcription of the target genes by associating with LEF/TCF transcriptional factor when Wnt signaling is activated. In addition, transcriptional activation is influenced by the interaction of βcatenin with the histone acetyltransferase CBP, the chromatin remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg) [14] . The Wnt/β-catenin pathway is vital for hair follicle development during gestation and growth cycling during postnatal life. The Wnt/β-catenin signaling is indispensable for the initiation and maintenance of the hair follicle placodes by cooperation with EDA/EDAR/NF-κB signaling [15][16][17] . Transient activation of β-catenin in telogen follicles triggers the subsequent anagen, indicating Wnt/β-catenin signaling is crucial for the hair follicle postnatal growth cycling [18] . Recent research has revealed how Wnt/β-catenin signaling can maintain hair-inducing activity of the dermal papilla, the dermal organizing center, in anagen using a skin reconstitution assay [19,20] . Importantly, Wnt/β-catenin signaling is involved in both hair follicle formation and hair growth in the adult.
MicroRNAs (miRNAs), small non-coding RNAs, are single strand molecules of about 22 nt in length processed by Dicer from hairpin precursors and exert their function by targeting mRNA for cleavage or inhibiting translation [21][22][23][24] . Most of miRNAs are highly conserved and have been implicated in participating in every aspect of biological processes, including embryonic development, apoptosis and proliferation, myogenesis and cardiogenesis [25,26] . The analysis of the microRNA expression profiling in skin and hair or feather follicles has been used to discover many important miRNAs related to hair follicle development and growth, especially in sheep, goat and duck [27][28][29][30] . It has been reported that the miRNA, miR-24 promotes hair follicle development by directly targeting Tcf-3 and miR-31 regulates anagen progression and hair follicle formation via inhibiting a number of growth regulatory molecules and cytoskeletal proteins [31,32] . Although the role of miRNAs in hair follicle development and growth has recently been explored, the specific molecular mechanisms involved are still unknown. In our previous study, we found that miR-148b was highly expressed in anagen compared with telogen [33] . MiR-148b belongs to the miR148/152 family and is involved in the regulation of development of disease and normal tissues [34] . It has been reported that miR-148b is required for osteogenesis [35] . During adipogenesis the expression of miR-148b is upregulated, which supports a potential role of miR-148b in this process [36] . CaMKIIα can be downregulated by miR-148b, which suppresses the innate response and Ag-presenting capacity of DCs [37] . Given the high expression of miR-148b in plasma from breast cancer cases, it can be used as a potential biomarker for breast cancer detection [38] . However, the specific function and underlying molecular mechanism of miR-148b in hair follicle growth remain unknown.
In this study, we aimed to investigate the role of miR-148b in the hair follicle growth cycle. First, the expression pattern of miR-148b in anagen and telogen was detected by qRT-PCR. Also, we explored the effect of miR-148b on the proliferation of hair follicle cells and subsequently investigated the signal pathway by which miR-148b functions. Finally, the targets of miR-148b were predicted and confirmed by a Dual Luciferase system and Western blotting.

Animal materials and sample collection
The wool bulbs in different phases (anagen, catagen, telogen) were plucked from three 2-year-old healthy Tibetan sheep supplied by the Agricultural and Animal Husbandry College of Tibet and immediately homogenized in TRIzol regent (Invitrogen, Carlsbad, CA, USA). After centrifuging at 12000 r$min -1 at 4°C for 10 min, the supernatant was preserved at -80°C.

Cells and cell culture
Human hair dermal papilla cells (HHDPC) and human hair germinal matrix cells (HHGMC) (ScienCell, San Diego, CA, USA) were cultured with mesenchymal stem cell medium, and maintained in a humidified incubator at 37°C supplemented by 5% CO 2 . HEK293 and MCF7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) at 37°C in 5% CO 2 .

Real-time quantitative PCR
Total mRNA and miRNA were extracted from cells and tissues with TRIzol reagent (Takara Bio In., Dalian, China) and treated with DNase I (Takara Bio In., Dalian, China), the concentration and quality of RNA was assessed through the NanoDrop 2000 and denaturing gel electrophoresis. Reverse transcription was conducted with reverse transcription kit (Takara Bio In., Dalian, China). Random primers, oligo (dT) 12-18 or miRNA specific primers were applied to initiate cDNA synthesis. The real-time quantitative PCR reaction was performed in a LightCycler 480 II (Roche, Basel, Switzerland) system using LightCycler 480 SYBR Green I Master (Roche). The sequences of the miRNA-specific and detection primers are listed in Table 1.

Dual-Luciferase reporter assays
The 3′UTR of NFAT5, Wnt10b housing the miR-148b targeting sequence was cloned into the psiCHECK-2 Dual-Luciferase reporter plasmid (Promega, Fitchburg, WI, USA). NFAT5 3′UTR was amplified with forward primer: CCGCTCGAGCATAGGCAAACTGTTGGGAGC and The experiments were performed in accordance with the 1996 Guide for the Care and Use of Laboratory Animals [39] . The protocols were approved by the Hubei Province for Biological Studies Animal Care and Use Committee and the Ethics Committee of Huazhong Agricultural University.

MiR-148b is significantly upregulated during the wool bulb anagen of Tibetan sheep
Previously, we found through Solexa sequencing that the expression of miR-148b in the anagen of wool bulbs was over 5 times the level in telogen, which indicates a potential role of miR-148b in the wool cycle in Tibetan sheep [28] . To confirm the expression profile of miR-148b, we collected the hair bulbs of Tibetan sheep in both anagen and telogen to analyze the expression of the miR-148b in hair bulbs by qRT-PCR. The data showed that the expression of miR-148b was upregulated in the anagen and downregulated in the telogen of Tibetan sheep wool bulb (Fig. 1).

Overexpression of miR-148b promotes the proliferation of the hair follicle cells in vitro
We evaluated the role of miR-148b in the proliferation of human dermal papilla cell and human germinal matrix cells, using the xCELLigence system to measure the proliferation of the dermal papilla cells. The data showed that the growth rate of the dermal papilla cells transfected with miR-148b was higher than when transfected with the NC sequence (Fig. 2a). Subsequently EdU staining was performed to confirm the proliferation tendency of the human dermal papilla cells and human germinal matrix cells. Similarly, the amount of cells that were in a proliferative state (EdU-positive cells) was significantly increased in miR-148b transfected cells compared with NC transfected cells (Fig. 2b). These data suggest that miR-148b can promote the proliferation of hair follicle cells.

miR-148b induces the activation of the Wnt signal pathway
To investigate the potential mechanism required for the effect of miR-148b on hair follicle cell proliferation, a Wnt signal reporter system was exploited to detect the activity of the Wnt signal pathway in HEK293 cells via luciferase reporter assay. The luciferase activity in HEK293 cells cotransfected with miR-148b, TOPFlash and PRL-TK was significantly higher than in cells cotransfected with the NC sequence, TOPFlash vector and PRL-TK. However, there was no significant difference between them using the TOPFlash instead of FOPFlash (Fig. 3a). Thus, the result indicates that overexpression of miR-148b activates the Wnt signal pathway. Subsequently, qRT-PCR was conducted to analyze the effect of miR-148b on cell cycle relevant factors in the Wnt signal pathway in human dermal papilla. β-catenin, a vital factor in the Wnt pathway, was upregulated in miR-148btreated HHDPC (Fig. 3c). Furthermore, compared to the NC transfected HHDPC, the downstream genes of the Wnt pathway, such as cycD, c-jun and Peroxisome Proliferator-Activated Receptor Delta (PPARD) that are directly  related to cell proliferation, were positively regulated significantly in HHDPC with miR-148b overexpression (Fig. 3d). Thus, collectively,overexpression of miR-148b upregulates the expression of genes, including cycD, c-jun and PPARD, through the activation of the Wnt signal pathway.

NFAT5 and Wnt10b were direct targets of miR-148b
To understand the mechanism by which miR-148b functions, we predicted the potential target genes of miR-148b by the bioinformatic tool, Targetscan. Of the target genes, NFAT5 and Wnt10b participated in Wnt signaling and were related to cell proliferation. The effect of miR-148b on NFAT5 and Wnt10b was validated by a Dual-Luciferase reporter system in HEK293 cells transfected with constructs containing the corresponding 3′UTR. We found that in comparison to NC-treated HEK293 the luciferase activity was significantly inhibited in HEK293 cells transfected with miR-148b mimics, indicating that miR-148b reduced the expression of NFAT5 and Wnt10b by binding to their 3′UTRs (Fig. 4a). In addition, Western blotting was performed with the protein extracted from MCF7 overexpressing miR-148b/NC to confirm the results found above. Consistently, there was a significant decrease of NFAT5 protein in MCF7 cells with miR-148b mimics (Fig. 4b, Fig. 4c). Together, these results revealed that miR-148b can target NFAT5 and Wnt10b to indirectly mediate the activity of the Wnt signal pathway or cell proliferation.

Discussion
Hair follicles are continually regenerating miniorgans undergoing the processes of proliferation, differentiation and apoptosis throughout an animal's lifetime. Anagen of the hair follicle is characterized by the activation of hair follicle stem cell clustering in the hair bulge area and subsequently the proliferation of the matrix cells, known as transit-amplifying cells, before differentiation [40] . MiR-NAs control expression of protein coding genes by translational inhibition and mRNA destabilization to regulate the biological process [25] . Dicer required for processing of miRNA and assembling into the RNAinduced silencing complex is essential for maintenance of the normal hair follicle, which indicates the necessity for miRNA in development and growth of hair follicles [41] . MiR-148b was found to participate in osteogenesis, adipogenesis and be dysregulated in many cancers [35,36] . It was also shown that miR-148b is significantly upregulated in wool follicle anagen of Tibetan sheep. Fig. 3 miR-148b activated Wnt signal pathway. (a) miR-148b mimics or NC were cotransfected with TOPFlash and PRL-TK plasmid or FOPFlash and PRL-TK plasmid, respectively, into HEK293 cells. Normalized luciferase activity was collected; (b) after transfection with miR-148b to HHDPC, cells were analyzed by qRT-PCR for detection of miR-148b. The expression of miR-148b was normalized to U6; (c) the expression of β-catenin in HHDPC overexpressing miR-148b was determined through qRT-PCR, and its expression was corrected to GAPDH; (d) the factors downstream of Wnt signal pathway, cycD, c-jun and PPARD, were detected by qRT-PCR. Their expression levels were normalized to GAPDH. Results are presented as meanAES.E.M (three independent replicates per group). *, P < 0.05; **, P < 0.01; ***, P < 0.0001.
Subsequently, we demonstrated that miR-148b promotes the proliferation of HHDPC and HHGMC. In contrast, Zhang et al. reported that miR-148b suppressed hepatocelluar carcinoma growth by targeting the Wnt/β-catenin pathway [42] . Likewise, there are differing results for the expression of miR-148b in specific cancers, such as downregulation in colorectal cancer, gastric cancer and breast cancer, conversely upregulation in ovarian carcinoma [43][44][45][46] . The conflicting results may be due to the difference in functions of miR-148b in various types of tissues. Therefore, miR-148b probably regulates proliferation of the hair follicle cells, especially hair matrix cells, to increase the growth rate of the hair follicle.
The Wnt pathway, initiated after the combination of Wnt family members and respective receptors/coreceptors, is needed for both development and homeostasis in tissues by bridging short range cell-cell communication [13,47] . The canonical Wnt/β-catenin pathway has been identified to contribute to development and growth of the hair follicle. β-catenin, a critical factor in the canonical Wnt pathway, is necessary for the induction and morphogenesis of the hair follicle by determining the fate of embryonic epidermis [48,49] . β-catenin in epidermis and hair follicle dermal papilla is required to promote the proliferation of progenitors to induce or maintain the anagen of the hair follicle [50][51][52] . When the Wnt/β-catenin pathway was activated, the luciferase activity of TOPFlash containing T cell binding site (Tcf) would be significantly increased compared with that in FOPFlash with mutated Tcf binding site. As expected, our results from the TOPFlash luciferase reporter system indicated that miR-148b could enhance the activity of the Wnt/β-catenin pathway, revealing a potential role for miR-148b in hair follicle growth cycling. Then, factors functioning in Wnt/β-catenin pathway, including β-catenin, cycD, c-jun and PPARD, were found to be upregulated in HHDPC overexpressing miR-148b, further substantiating that miR-148b can positively regulate the Wnt/β-catenin pathway. Previous reports indicate that βcatenin signaling in both epidermis and dermal papilla is essential for maintenance of proliferation in anagen hair follicles [49,51] . Therefore miR-148b was considered to stimulate the progress of the hair follicle anagen by means of activating the Wnt/β-catenin pathway to promote proliferation in vivo.
To reveal the underlying mechanism used by miR-148b, putative target genes (Wnt10b and NFAT5) of miR-148b Fig. 4 NFAT5 and Wnt10b were identified as the targets of miR-148b. (a) The 3′URT of NFAT5 and Wnt10b were inserted into the dualluciferase reporter vector psi-CHECK2 at the end of the Renllia luciferase gene (hRluc). miR-148b mimics or NC were cotransfected with psi-CHECK2 containing 3′UTR mentioned above, into HEK293 cells. The constitutive firefly luciferase (hluc + ) expression was used as an internal normalized control, and normalized Renllia luciferase activity was assayed; (b) NFAT5 protein expression in MCF7 cells was determined after transfection with miR-148b into the MCF7 cells for 36 h. Comparable GAPDH levels served as a loading control during western blotting; (c) results of NFAT5 protein expression in MCF7 cells are presented as meanAES.E.M (three independent replicates per group). *, P < 0.05; **, P < 0.01. were indentified. The luciferase reporter results revealed miR-148b could bind to the 3′UTRs of the two genes. In addition, the expression of the NFAT5 protein was significantly repressed by overexpression of miR-148b.
Our results indicate that miR-148b can bind to Wnt10b and NFAT5 mRNAs to suppress their translation directly.
The Wnt/β-catenin pathway engages in wide range of biological events, including cell proliferation, terminal differentiation and maintenance or activation of stem cells [13] . However, the precise function of Wnt/β-catenin has yet to be fully known. Wnt10b promotes differentiation in hair follicles to influence hair shaft growth [53,54] . Also, it was reported that Wnt10b suppressed proliferation and promoted the differentiation in epithelial stem/progenitor cells and melanocytes in the skin through activating canonical Wnt signaling [55,56] . MiR-148b may promote the growth rate of hair follicle by downregualtion of Wnt10b in hair follicle cells that rapidly proliferates in anagen. βcatenin contains several functional domains, including the N-terminal domain responsible for β-catenin stability, the central armadillo domains that bind to TCF/LEF and Ecadherin and C-terminal domain that interacts with transcriptional mediator complex [54,57] . Considering various aspects of Wnt/β-catenin signaling, not only are βcatenin nuclear localization and its interaction with TCF important, but also the combination of β-catenin and transcriptional mediators plays a crucial role [58,59] . NFAT5 can interact with the C-terminal transactivation domain of β-catenin to block its interaction with CBP to inhibit CBPmediated β-catenin acetylation and ultimately inhibit the canonical Wnt pathway [58] . The interaction between NFAT5 and β-catenin can be counteracted by miR-148b and then leads to acetylation of β-catenin. Subsequently, the acetylated β-catenin activates the Wnt/β-catenin pathway to increase the expression of downstream genes, cycD, c-jun and PPARD which are related to the cell cycle. Although, the role of NFAT5 in hair follicles still remains unclear, we have provided evidence that miR-148b targets Wnt10b and NFAT5 to regulate the proliferation of hair follicle cells.

Conclusions
This study found that miR-148b was highly expressed in the anagen of hair follicle and could promote proliferation of hair follicle cells. In addition, we provided data that indicates miR-148b could activate Wnt/β-catenin signaling by upregulating the expression of b-catenin, cycD, c-jun and PPARD, which may be mediated by targeting NFAT5 and Wnt10b. These results suggested that miR-148b might be important for increased proliferation of hair follicle in anagen.