LncRNA-XIST promotes dermal papilla induced hair follicle regeneration by targeting miR-424 to activate hedgehog signaling

Cellular Signalling 72 (2020) 109623

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LncRNA-XIST promotes dermal papilla induced hair follicle regeneration by targeting miR-424 to activate hedgehog signaling

T

Bo-Jie Lina,1, Jiang-Ying Zhub,1, Jun Yec, Si-Ding Lua, Ming-De Liaoa, Xu-Chang Menga, ⁎ Guo-Qian Yina, a

Department of Plastic and Aesthetic Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Province, PR China Academy of Humanities and Social Sciences, Guangxi Medical University, Nanning 530021, Guangxi Province, PR China c Department of Emergency Surgery, The Affiliated Zhuzhou Hospital Xiangya Medical College CSU, Zhuzhou 412007, Hunan Province, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: lncRNA-XIST miR-424 Hedgehog signaling pathway Dermal papilla Hair follicle regeneration

Background: Alopecia is a highly prevalent disease characterizing by the loss of hair. Dermal papilla (DP) cells are the inducer of hair follicle regeneration, and in vitro three-dimensional (3D) culturing DP cells have been proven to induce hair follicle regeneration. However, the molecular mechanisms behind the regulation of 3D culturing DP cells remain unclear. Methods: 3D-cultivated DP cells were used as in vitro cell model. DP sphere xenograft to nude mice was performed for in vivo study of hair follicle regeneration. qRT-PCR, Western blotting, and immunofluorescence were used for detecting the level of XIST, miR-424 and Hedgehog pathway-related proteins, respectively. H&E staining was used to examine hair neogenesis. Cell viability, proliferation and ALP activity were measured by MTT, CCK-8 and ELISA assays, respectively. Luciferase assays were used for studying molecular regulation between XIST, miR-424 and Shh 3′UTR. Results: XIST and Shh were up-regulated, and miR-424 was down-regulated in 3D DP cells. Molecular regulation studies suggested that XIST sponged miR-424 to promote Shh expression. Knockdown of XIST suppressed DP cell activity, cell proliferation, ALP activity and the expression of other DP markers by sponging miR-424. Knockdown of XIST suppressed Shh mediated hedgehog signaling by targeting miR-424. Moreover, the knockdown of XIST inhibited DP sphere induced in vivo hair follicle regeneration and hair development. Conclusion: XIST sponges miR-424 to promote Shh expression, thereby activating hedgehog signaling and facilitating DP mediated hair follicle regeneration.

1. Introduction Alopecia is a disease characterized by the loss of hair [1], and brings high psychological burdens for the patients, no matter male or female [2]. The causes of hair loss are complicated, leading to different types of alopecia, including androgen alopecia [3], alopecia areata [4] and scarring alopecia [5], etc. The occurrence rate of alopecia is very high. For example, estimated that the lifetime incidence rate of alopecia areata is approximately 2% worldwide [6]. Due to the lack of understanding for pathophysiological and molecular mechanisms, there are still no efficient therapeutic agents for the prevention and treatment of alopecia. The structure of hair follicle consists of hair shaft (HF), inner root

sheath (IRS), and outer root sheath (ORS), all of which are formed by keratinocytes [7]. There is a special group of mesenchymal cells located at the basal of follicle, termed dermal papilla cells, and formed the dermal papilla (DP) of hair follicle [8]. Recent studies focused on the interactions of DP and epidemic keratinocytes, suggested that DP cells contribute greatly to the regulation of hair follicle regeneration [9]. Previous studies demonstrated that DP guided the formation of the epithelial part of hair follicle. Additionally, DP produces factors to regulate the initiation of anagen stage of hair follicle cycle by stimulating hair germ and quiescent bulge stem cells [10]. Therefore, DP exerts positive functions for hair follicle regeneration. Additionally, the in vitro three-dimensional (3D) culturing of DP cells has been developed recently, this method has been proven to avoid the loss of DP activity

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Corresponding author at: Department of Plastic and Aesthetic Surgery, The First Affiliated Hospital of Guangxi Medical University, No.6 Shuangyong Road, Nanning 530021, Guangxi Province, PR China. E-mail address: [email protected] (G.-Q. Yin). 1 These are co-first authors. https://doi.org/10.1016/j.cellsig.2020.109623 Received 31 December 2019; Received in revised form 30 March 2020; Accepted 30 March 2020 Available online 31 March 2020 0898-6568/ © 2020 Elsevier Inc. All rights reserved.

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regulating hair follicle regeneration remains unclear. A recent report indicated that a Turner syndrome patient with the lack of XIST gene unexpectedly presented alopecia universalis phenotype, suggesting that XIST may be associated with hair loss [32]. Due to the fact that the disorder of DP mediated hair follicle regeneration leads to alopecia, we speculated that XIST may have a role in the regulation of DP and hair follicle regeneration. In the present study, based on the predictions of lncRNA-miRNA and miRNA-mRNA interactions from databases, we investigated the roles of lncRNA-XIST and miR-424 in the regulation of DP mediated hair follicle regeneration. We identified that XIST/miR-424/Shh axis is a critical pathway in regulating DP cells and the consequent hair follicle regeneration. This new mechanism provides deeper insight into the regulation of DP mediated hair folicle regeneration, and XIST may be a new therapeutic target for the treatment of alopecia.

after passages, compared to two-dimensional (2D) DP culturing [11]. Thus is considered as a potential tissue engineering method for the treatment of alopecia. However, the molecular mechanisms for the maintenance of DP activity by 3D DP culturing remain to be investigated. Sonic hedgehog (Shh) signaling is one of the fundamental signaling pathways that contribute to hair follicle development and follicle bulge stem cell maintenance [12]. Shh signaling can be activated by the binding of Hedgehog ligands to their receptors, including Patched (PTCH) 1 or 2, which are 12-pass transmembrane-spanning receptors. After the bind of ligands and receptors, cell signaling transduces to smoothened (SMO) protein and then activate glioma-associated (GLI) protein, which participates in the regulation of target gene expression [13]. Previous studies demonstrated that Shh signaling regulates the development of hair follicle by functioning in both epithelium and mesenchyme [14,15]; thereof, it regulates both keratinocytes and dermal papilla cells. Knockout of Shh gene in mice resulted in a reduction of keratinocyte proliferation, and smaller dermal papilla, compared to WT mice [15]. Additionally, knockdown of SMO specifically in dermal cells resulted in the loss of dermal papilla precursor cells, dermal cells condensate and resulted in failure of hair follicle development [16]. Recent studies from Lim, et.al revealed that Shh stimulates hair follicle neogenesis by creating inductive dermis during murine skin wound healing [17]，suggesting that Shh signaling is crucial for hair regeneration. MicroRNAs (miRNAs) are a class of small non-coding RNAs which play a key role in the regulations of cell proliferation, cell differentiation and cell survival [18]. MiRNAs exert their function by binding to the 3′ untranslational region (3′UTR) of complementary mRNAs and lead to the translational inhibition or degradation of their target mRNAs [18]. Recent studies identified that a variety of miRNAs are involved in the regulation of hair follicle regeneration [19]. For example, miR-214 regulates hair follicle regeneration by modulating Wnt signaling [20]. MiR-24 regulates hair follicle regeneration by targeting Tcf-3 [21]. However, few of the miRNAs are found to regulate dermal papilla. A recent study indicated that, miR-424, which are included in miR-15 family (contains miR-15a, 15b, 16, 195, 424 and 497) targets Shh signaling pathway during postnatal development of porcine intestine [22]. Thus, we prospected that miR-424 may target to Shh signaling in hair follicle as well. However, the exact role of miR-424 in the regulation of DP cells and hair follicle regeneration is still unknown and to be elucidated. Long non-coding RNAs (lncRNAs) are broadly defined as noncoding RNA molecules longer than 200 nucleotides [23]. Similar as mRNAs, lncRNAs contain a 5′ 7-methylguanosine cap and a 3′ poly(A) tail, however, they lack coding capacity [23]. LncRNAs exert functions via different mechanisms, including regulating chromatin remodeling, transcriptional and post-transcriptional regulation and act as competing endogenous RNA (ceRNA) to sponge miRNAs, etc. [24]. The ceRNA mechanism plays a crucial role for lncRNAs in the regulation of gene expression. MiRNAs inhibit the expression of target genes by binding to their transcripted mRNAs and facilitate mRNA degradation. LncRNAs function as ceRNA to compete this function of miRNAs by direct lncRNA-miRNA bindings, thus lead to blockage of miRNAs mediated inhibition of gene expression. This lncRNA derived miRNA inhibition is termed as “sponge”. For example, lncRNA AC073284.4 suppresses epithelial-mesenchymal transition (EMT) by sponging miR-18b-5p in paclitaxel-resistant breast cancer cells [25]. LncRNA FOXP4-AS1 is activated by PAX5 and promotes the growth of prostate cancer by sponging miR-3184-5p to upregulate FOXP4 expression [26]. To date, lncRNAs are found to be involved in the regulation of hair follicle, although still very rare [27–30]. Therefore, the potential functions of lncRNAs in regulating hair follicle development or cycling remain to be identified. X-inactive-specific transcript (XIST) is one of the first lncRNAs discovered in mammals and plays an essential role in X chromosome inactivation [31]. However, the exact role of XIST in

2. Materials and methods 2.1. Isolation of DP cells To isolate DP cells, human scalp samples were collected from face lifting surgery patients who obtained informed consent. Micro dissected DP from scalp samples were cultured in the Dulbecco's modified Eagle's medium (DMEM, Gibco, NY) supplemented with 1% (v/v) penicillinstreptomycin and 20% (v/v) fetal bovine serum (FBS, Gibco) in a humidified 5% CO2 incubator at 37 °C. When DP cells reached 70% confluent, a subculture was carried out. Cell culturing medium changed to DMEM supplemented with 10% FBS after 7 days of cultivation. 2.2. 2D and 3D culturing of DP cells 2D and 3D culturing of DP cells were performed following the previous report [11]. Briefly, cells were digested by trypsin and plated in either 35-mm dishes for 2D cell culturing, or in hanging drop cultures for 3D culturing using hanging drop plates (3D BiomatricInc., Michigan, USA). 10 μL of hanging drops consisted of 3000 cells in DMEM (containing 10% FBS) were used for 3D culturing. Cultures were maintained for between 30 and 72 h. 2.3. Gene knockdown or gene overexpression For the knockdown of XIST, a XIST shRNA was constructed, and packed by lenti-virus (customized ordered by GenePharma, Shanghai, China). For the overexpression or inhibition of miR-424, cells were transfected with miR-424 mimics or miR-424 inhibitor. MiR-424 mimics and miR-424 inhibitor were customized ordered by GenePharma (Shanghai, China). Then these constructs or RNAs were transfected into cells using Lipofectamine 2000 (Invitrogen) according with the product's protocol. After 48 h of transfection, the cells were harvested for subsequent qRT-PCR detection. Due to the fact that miR-424 and miR424 mimics have consistent sequences that can be probed by qRT-PCR primers, thus miR-424 mimics was also detected. 2.4. Immunofluorescence Cells were seeded into 8-chamber culture slides (BD Falcon, Franklin Lakes, NJ), with a density of 5 × 103 cells per chamber. The next day, cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Next, cells were permeabilizated with 0.1% Sodium Citrate plus 0.1% Triton X-100. Cells were incubated with anti-Ki-67 (1:500, Abcam) antibody, or anti-Shh (1:500, Abcam) for 2 h at room temperature. The cells were then washed with cold PBS three times and incubated with Alexa 488 or Alexa 594-labeled anti-rabbit secondary antibody (1:800) (Invitrogen) at room temperature for 1 h. Nucleus were stained with DAPI (SigmaAldrich). The cells were examined by fluorescence microscopy 2

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syntheized by reverse transcription with M-MLV reversed transcriptase (ThermoFisher Scientific). cDNA was diluted to be 20 ng/mL and applied for qRT-PCR. The qRT-PCR reactions were carried out in ABI 7500 real time PCR system (Applied Biosystems), by using the SYBR Green PCR Master Mix reagents (Takara, Dalian, China). The PCR cycles were composed of 50 °C for 2 min followed by an initial denaturation step at 95 °C for 10 min, 45 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The experiments were carried out in triplicate for each data point. The relative quantification in gene expression was determined using the 2-ΔΔCt method [34]. The primers used in this study include: XIST forward 5′-ACGTCGGCTATCAGAGCAAG-3′, reverse 5′-AGGCCTTCGGTC CAATTCAG-3′; miR-424 forward 5′-CGGCAGCAGCAATTCATGT-3′, reverse 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACG ACTTCAAA-3′; Shh forward 5′-AAAAGCTGACCCCTTTAGCC-3′, reverse 5′-GCTCCGGTGTTTTCTTCATC-3′; U6 forward 5′-CTCGCTTCGGCAGC ACA-3′, reverse 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH forward 5′-CCAGGTGGTCTCCTCTGA-3′, reverse 5′- GCTGTAGCCAAATCGT TGT-3′. U6 and GAPDH were used as internal control.

(Olympus America Inc., Center Valley, PA). 2.5. MTT assay DP cells were seeded in hanging drop cultures for 3D culturing using hanging drop plates (3D BiomatricInc., Michigan, USA) and incubated for indicated durations. Each well of the cells was added with 20 μL MTT (5 mg/mL) and incubated for 3 h. Next, the culture medium containing MTT solution was removed and the Formazan crystals were dissolved in 100 μL of DMSO. Absorbance was read with a Microplate Reader (Thermo Fisher Scientific) at 490 nm. 2.6. CCK-8 assay For Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) assay, DP cells were seeded into 96-well plates. After cultured at 37 °C for indicated time, 100 μL of CCK-8 solution was added into each well and incubated for another 2 h. Finally, the absorbance was determined at 450 nm using a microplate reader (Bio-Rad Laboratories, USA).

2.12. Western blot analysis

2.7. ELISA

Total proteins were extracted by cell lysis buffer (Tris-HCl, PH 8.0, 400 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM Na pyrophosphate, 1% Triton X-100, 10% glycerol), with the supplement of protease inhibitors (Thermofisher). The concentration of protein was measured by a BCA kit (Piere, Rockford, IL). The proteins were loaded onto 12% SDS-PAGE gel and were transferred to PVDF membranes (Millipore, Billerica, MA). Membranes were then incubated with primary and secondary antibodies. Protein signals were detected via enhanced chemiluminescence (ECL) method. The primary antibodies used in this study include antiALP (1:1500, Abcam), anti-NCAM (1:1000, Abcam), anti-Versican (1:1000, Abcam), anti-α-SMA (1:1000, Abcam), anti-Shh (1:1000, Abcam), anti-GAPDH (1:3000, Abcam), anti-Ptch1 (1:1000, Abcam), anti-Gli1 (1:800, Abcam) and anti-Gli2 (1:800, Abcam). All the primary antibodies were incubated overnight at 4 °C. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h at room temperature. Then the enhanced chemiluminescence kit (Thermo Fisher Scientific, USA) was applied to detect the reacted protein bands.

After 3D culturing, cells and medium were collected and centrifuged at 1000g at 4 °C and supernatant was collected for assay. The detection of secreted form of ALP was performed by an ALP ELISA Kit (Biocompare) according to the manufacturer's instructions. 2.8. Luciferase reporter assay To examine the relationship between miR-424 and XIST, cells were transiently co-transfected with pGL3-XIST and miR-424 mimics/miR424 inhibitor, or co-transfected with pGL3-XIST mutant and miR-424 mimics/miR-424 inhibitors. To examine the relationship between miR424 and Shh 3’UTR, cells were transiently co-transfected with pGL3Shh 3’UTR-Wildtype and miR-424 mimics/miR-424 inhibitor, or pGL3Shh 3’UTR-Mutant and miR-424 mimics/miR-424 inhibitors. Luciferase assay was performed by Dual-Luciferase® Reporter Assay System (Promega), following the instructions of manufacturer.

2.13. Statistical analysis 2.9. In vivo hair formation Data are expressed as mean ± standard deviation (SD) values. Comparisons between more than two groups were analyzed by one-way ANOVA followed by Tukey post hoc tests. Comparisons between two groups were made with two-tailed t-tests. Statistical analysis was performed using SPSS 10.0 for Windows. P < .05 was considered statistically significant.

As previous described, patch assay was used for evaluating the capability of DP spheres induced hair formation in nude mice [33]. Nude mice were purchased from SJA Laboratory Animal Co., Ltd. (Hunan, China). After anesthetized by isoflurane (Sigma-Aldrich), nude mice were operated and form a small full-thickness wound on the lateral dorsal skin. 1 × 106 freshly isolated epidermal cells from new born C57BL/6 were combined with a total of 60 P8-DP spheroids that harvested from hanging drop array plates and injected into the hypodermis area with pipette. The skin was sutured after injection and then the fullthickness tissue was harvested after sacrifice of mice at 4 weeks after the implantation.

3. Results 3.1. Gene expression of XIST, miR-424 and the activation of hedgehog signaling in 3D culturing of DP cells According to our previous reports, 3D culturing of dermal papilla cells can mimic the in vivo dermal papilla in hair follicle and presented much better effects than 2D culturing [11]. In this study, we used this in vitro DP model with 3D culturing of DP cells. Firstly, we examined the expression of XIST, miR-424 and Shh. As shown in Fig. 1A, qRT-PCR results indicated that, the gene expression of XIST was stimulated in 3D culturing group, compared to 2D culture group and freshly isolated DP cells. Oppositely, the expression of miR-424 was down-regulated in 3D culturing (Fig. 1B). The expression of Shh was stimulated (Fig. 1C), similar as the pattern of XIST expression, compared to 2D culture group and freshly isolated DP cells. Additionally, the expression of those genes in freshly isolated DP cells was consistent with the cultivated 2D DP cells, and exhibited no significant difference (Fig. 1A–C). Next, we

2.10. H&E staining Freshly isolated skin tissues were collected and fixed in 4% paraformaldehyde (PFA) in PBS overnight, followed by embedding and sectioning (5 μm) in paraffin. Hematoxylin and eosin (H&E) staining were performed using H&E Staining Kit (Abcam) according to the manufacturer's instructions. 2.11. qRT-PCR The gene expression of XIST, miR-424 and Shh were evaluated by qRT-PCR. Total RNA was extracted by Trizol (Invitrogen). cDNA was 3

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Fig. 1. Expression of XIST, miR-424 and Shh in 3D culturing DP cells. Relative gene expression of XIST (A), miR-424 (B) and Shh (C) in 2D and 3D culturing of DP cells. The protein level of Shh in 2D and 3D culturing of DP cells was measured by immunofluorescence (D). Western blotting for analysis of ALP, NCAM, Versican, αSMA, Shh, Ptch1, Gli1 and Gli2 protein level in 2D and 3D culturing of DP cells (E), and the quantification of Western blotting results (F). All the results were shown as mean ± SD. * p < .05 and ** p < .01.

3.2. XIST promoted Shh expression by sponging miR-424

confirmed the expression of Shh protein via immunofluorescence method. As shown in Fig. 1D, notably stronger expression of Shh expression was found in the 3D culturing DP cells, compared to 2D culturing and freshly isolated DP cells. To confirm the effect of mimicking in vivo DP, we evaluated the DP markers in 3D culturing. Western blotting results indicated that the protein level of DP markers, including ALP, NCAM, Versican and α-SMA were all elevated in 3D culturing (Fig. 1E&F). Consistent with the qRT-PCR results, Shh protein level was up-regulated in 3D culturing (Fig. 1E&F). Additionally, protein level of the hedgehog signaling components, including Ptch1, Gli1 and Gli2, were all up-regulated in 3D culturing (Fig. 1E&F). These results suggested that expression of XIST and Shh were up-regulated, but miR-424 was down-regulated in 3D DP spheroid. Additionally, Hedgehog signaling was activated in this in vitro 3D cell model.

The negative correlation between the expression of XIST and miR424 in 3D DP culturing implied that the two RNAs may have negatively regulatory relationship. To confirm this hypothesis, gene knockdown of XIST and transfection of miR-424 mimics or inhibitor were performed. As shown in Fig. 2A, XIST was knockdown by the transfection of shRNA-XIST. The RNA level of miR-424 was sharply stimulated by the knockdown of XIST. Furthermore, the level of Shh was found to be reduced by the knockdown of XIST (Fig. 2A). Next, the level of miR-424 was confirmed to be stimulated by the transfection of miR-424 mimics, however, was reduced significantly by miR-424 inhibitor (Fig. 2B). These results were consistent with our prospects, since the miR-424 mimics had same sequences with native endogenous miR-424, and both miR-424 and miR-424 mimics could be detected by qRT-PCR primers. This phenomenon was also consistent with previous reports [35]. Importantly, the expression of Shh was found to be suppressed by miR-424

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Fig. 2. XIST promoted Shh expression by sponging miR-424. After treatment of shNC or shXIST in 3D DP cells, the expression level of XIST, miR-424 and Shh was determined by qRT-PCR (A). After treatment of mimics NC, miR-424 mimics, inhibitor NC or miR-424 inhibitor in 3D DP cells, miR-424 and Shh expression were determined by qRT-PCR (B). Bioinformatics prediction of binding sites between XIST and miR-424 (C), between miR-424 and Shh 3’UTR (D) were shown. After treatment of mimics NC, miR-424 mimics, inhibitor NC or miR-424 inhibitor, XIST-WT or XIST-MUT mediated luciferase activity (E), and Shh 3’UTR mediated luciferase activity (F) was measured. All the results were shown as mean ± SD. * p < .05, ** p < .01 and *** p < .001.

proliferation of DP cells were reduced by the knockdown of XIST or the transfection of miR-424 mimics. Oppositely, XIST knockdown mediated suppression of cell viability or proliferation rate were reversed by transfection of miR-424 inhibitor, suggesting that XIST promoted cell viability and proliferation of DP cells by regulating miR-424 (Fig. 3A& B). These results were confirmed by the immunofluorescence assays. The expression of proliferation marker Ki-67 was reduced by the knockdown of XIST or the transfection of miR-424 mimics, however, was stimulated by miR-424 inhibitor (Fig. 3C). Collectively, these results suggested that knockdown of XIST suppressed the proliferation of DP cells by targeting miR-424.

mimics but was promoted by miR-424 inhibitor (Fig. 2B). Based on these results, we speculated that XIST may bind to miR-424 and inhibit its function, and miR-424 may bind to the 3’ UTR of Shh to suppress its expression. Next, bioinformatics analysis was used to predict these putative binding modes. As shown in Fig. 2C&D, we found the binding sites between XIST and miR-424, miR-424 and Shh in the database Starbase (http://starbase.sysu.edu.cn/index.php) and Targetscan database (http://www.targetscan.org/vert_71/), respectively. To confirm these putative regulations, luciferase assays were performed. Results indicated that luciferase activity mediated by wild type XIST was suppressed by miR-424 mimics, however, luciferase activity mediated by XIST mutant was not affected (Fig. 2E). Oppositely, miR-424 inhibitor stimulated the luciferase activity mediated by wild type XIST, but didn't impact the mutant one (Fig. 2E). These results suggested that XIST bound to miR-424 and acted as ceRNA to exert sponging effects. As shown in Fig. 2F, wild type Shh 3′UTR mediated luciferase activity was suppressed by miR-424 mimics, however, was stimulated by miR-424 inhibitor. Shh 3′UTR mutant mediated luciferase activity was not affected. These results suggested that miR-424 exerted negative regulations for Shh expression. Collectively, XIST may act as ceRNA to sponge miR-424 to relieve the miR-424 mediated suppression of Shh expression.

3.4. Knockdown of XIST suppressed ALP activity and expression of other DP markers by targeting miR-424 in 3D DP spheroid To confirm the role of XIST/miR-424 in regulating 3D DP spheroid, we evaluated ALP activity and the expression of other DP markers. As shown in Fig. 4A, the ALP activity of 3D DP spheroid was reduced by the knockdown of XIST, as well as the transfection of miR-424 mimics. However, ALP activity was promoted by transfection of miR-424 inhibitor, characterized by the result that miR-424 inhibitor reversed XIST knockdown mediated suppression of ALP activity (Fig. 4A). As shown in Fig. 4B, Western blotting results indicated that the protein levels of DP makers, including ALP, NCAM, Vesrican, α-SMA, were reduced by the knockdown of XIST or the transfection of miR-424 mimics (Fig. 4B&C). Oppositely, the transfection of miR-424 inhibitor was found to rescue the expression of DP markers, which was suppressed by XIST knockdown (Fig. 4B&C). These results suggested that knockdown of XIST suppressed ALP and other DP markers expression in DP

3.3. Knockdown of XIST suppressed DP cell activity and proliferation by targeting miR-424 To explore the role of XIST in the regulation of DP activity and cell proliferation, knockdown of XIST in 3D culturing of DP cells was performed. As shown in Fig. 3A&B, the relative cell viability or 5

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Fig. 3. XIST regulated viability and proliferation of 3D culturing DP cells via targeting miR-424. After treatment of shNC+inhibitor, mimics NC, shXIST+inhibitor NC, miR-424 mimics, or shXIST+miR-424 inhibitor in 3D DP cells, cell viability and proliferation was determined by MTT assay (A) and CCK-8 assay (B), and the expression of Ki-67 was detected by immunofluorescence (C). All the results were shown as mean ± SD. * p < .05 and ** p < .01.

B, miR-424 mimics reduced the protein level of Shh, Ptch1, Gli1 and Gli2. Contrast to this, the transfection of miR-424 inhibitor reversed XIST shRNA induced suppression of Shh, Ptch1, Gli1 and Gli2 protein level. These results suggested that the knockdown of XIST suppressed Shh expression and Shh mediated hedgehog signaling in 3D DP spheroid by targeting miR-424.

spheroid by targeting miR-424. 3.5. Knockdown of XIST suppressed Shh mediated hedgehog signaling by targeting miR-424 To ascertain whether XIST regulates DP activity by modulating Shh signaing, we examined the impacts of Shh expression and Hedgehog signal components by XIST knockdown. As shown in Fig. 5A&B, Western blotting results indicated that the knockdown of XIST resulted in a down-regulation of Shh protein level, suggesting that the expression of Shh was inhibited. Furthermore, the protein level of hedgehog associated components, including Ptch1, Gli1, Gli2 were suppressed. Similar results were found by the overexpression of miR-424, which was mediated by the transfection of miR-424 mimics. As shown in Fig. 5A&

3.6. Knockdown of XIST inhibited DP spheroid induced in vivo hair follicle regeneration and hair development To examine the function of XIST in the regulation of DP spheroid mediated hair follicle regeneration and hair development in vivo, a patch assay was performed. In this assay, DP spheroids with different treatments were injected to the hypodermis area of the skin of nude 6

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Fig. 4. Knockdown of XIST suppressed ALP activity and the expression of other hair-induction markers by targeting miR-424. ALP activity was measured by ELISA assay (A). The expression of hair-induction markers (ALP, NCAM, Versican, α-SMA) were determined by Western blotting (B), and the quantification of Western blotting results (C). All the results were shown as mean ± SD. * p < .05 and ** p < .01.

resulted in reduction of cell viability rate, cell proliferation rate, the expression of proliferation maker Ki-67, ALP activity and the expression of other DP markers, including NCAM, Versican and α-SMA. To our best of knowledge, our results provide the first expression profile of XIST in DP cells. Previous studies revealed that XIST play roles in regulating cell proliferation. For example, XIST was found to promote the proliferation of pancreatic cancer cells by targeting miR-133a to regulate EGFR expression [39]. In bladder cancer, XIST targets to miR-133a to stimulate cell proliferation [40]. In thyroid cancer, XIST targets miR-34a to promote MET-PI3K-AKT signaling so as to stimulate cell proliferation [41]. Thus, XIST is considered as a stimulator for cell proliferation and even an oncogene for tumor development. Our results indicated that XIST is a stimulator for the proliferation of 3D DP cells and is a critical activator for DP cell proliferation and activity. Our further in vivo studies demonstrated that knockdown of XIST in xenografted 3D DP spheroid led to attenuation of DP mediated hair follicle regeneration. Taken together of these in vitro and in vivo evidence, XIST exerts stimulative effects for DP cells proliferation and activity, as well as DP induced hair follicle regeneration. Next, we found that miR-424 mimics suppressed DP cell proliferation and activity in 3D DP culturing, and miR-424 inhibitor reversed XIST knockdown mediated suppression of DP proliferation and activity, demonstrating that XIST promoted DP proliferation and activity by targeting miR-424. Our study provided the first description about the function of miR-424 in regulating DP cells. According to previous reports, the function of miR-424 in regulating cell proliferation is complicated. In esophageal squamous cancer cells, miR-424 was found to promote cell proliferation by affecting cell cycling [42]. However, in hemangioma-derived endothelial cells, miR-424 suppressed its proliferation by targeting VEGFR2 [43]. Probably that the regulatory function of miR-424 for cell proliferation is diverse, and cell type dependent. For the first time, in the present study we identified that miR424 acts as a suppressor for proliferation and activity of DP cells, and can be suppressed by XIST via a ceRNA mechanism. Based on our findings, we prospect that the promotion of XIST or knockdown of miR-

mice. H&E staining results indicated that, nude mice skin with normal 3D DP spheroid injection led numerous growth of hair follicle, however, the one with shXIST DP spheroid injection resulted in significantly reduction of hair follicle (Fig. 6A). To validate the in vivo molecular mechanism, we investigated the activity of Hedgehog signaling and the expression of DP makers in the nude mice tissues. Compared to the control, the protein level of ALP, NCAM, Versican and α-SMA were all suppressed by shXIST in the xenografted skin tissue (Fig. 6B&C). Furthermore, the level of hedgehog signaling proteins, including Shh, Ptch1, Gli1 and Gli2 were found to be suppressed by shXIST in xenografted skin tissue of nude mice (Fig. 6B&C). These results fully demonstrated that XIST played a key role in 3D spheroid induced hair follicle regeneration by regulating hedgehog signaling pathway.

4. Discussion Alopecia is a highly prevalent disease and the prevention and treatment of alopecia is of great clinical importance [6]. Even though there are already reports to claim potential methods for the treatment, such as herbal alternatives [36], intralesional steroids and 5α-reductase inhibitors [1] and phosphodiesterase inhibitors [37], etc. However, there is still no efficient therapeutic agents for the treatment of alopecia in clinical market. The contribution of DP induced hair follicle regeneration to hair development has been validated recently [38]. Deeper insights into the molecular mechanisms of DP mediated hair follicle regeneration will facilitate drug development for the neogenesis of hair and the treatment of alopecia. In the present study, we investigated the role of XIST in the regulation of DP mediated hair follicle regeneration. According to previous reports, 3D culturing of DP cells presented advantages for mimicking the in vivo state of dermal papilla [11]. In this study, the 3D DP culturing was confirmed to be well-established, charactering by the significant high expression of the DP markers. Our results indicated that the expression of XIST was up-regulated, implying that XIST may play a positive role in DP regulation. Knockdown of XIST in 3D DP cells 7

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Fig. 5. Knockdown of XIST suppressed Hedgehog signaling by targeting miR-424. The protein level of Shh, Ptch1, Gli1 and Gli2 were measured by Western blotting (A), and the quantification of Western blotting results (B). The expression of Shh was determined by immunofluorescence (C). All the results were shown as mean ± SD. * p < .05 and ** p < .01.

miR-15 family (containing miR-15a, 15b, 16, 195, 424 and 497), is proposed to target hedgehog signaling pathway during postnatal development of porcine intestine [22]. However, this regulation is still not validated by strict experimental evidence. Our results indicated that the protein level of hedgehog signaling, including Shh, Ptch1, Gli1 and Gli2 were all elevated in 3D culturing DP cells, suggesting that Shh signaling was activated. Hedgehog signaling was suppressed by the knockdown of XIST and miR-424 mimics, but was promoted by miR-424 inhibitor.

424 in DP cells may be an efficient therapeutic strategy for DP activity and hair follicle regeneration, and finally hair neogenesis and alopecia treatment. Therefore, the overexpression of XIST or knockdown of miR424 in a novel gene therapy way may be a further direction for the treatment of alopecia. Previous studies demonstrated the critical role of hedgehog signaling in regulating DP mediated hair follicle regeneration in vivo [15,17]. A previous report indicated that miR-424, which is involved in 8

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Fig. 6. Knockdown of XIST suppressed 3D DP spheroid induced hair follicle regeneration in nude mice. 3D DP spheroid with or without XIST knockdown was injected to skin of nude mice. Formation of new-formed hair follicle was determined by H&E staining (A). Expression of ALP and DP mediated hair induction markers (NCAM, Versican and α-SMA) and Hedgehog signaling-related proteins (Shh, Ptch1, Gli1 and Gli2) were determined by Western blotting (B) and the relative quantification (C). All the results were shown as mean ± SD. * p < .05 and ** p < .01.

5. Conclusions

Results of the in vivo patch assay in nude mice also indicated that knockdown of XIST in the xenograft 3D DP spheroid resulted in suppression of hedgehog signaling, characterizing by the reduced level of Shh and other hedgehog signaling components. Previous studies indicated that XIST sponged various miRNAs to inhibit their activities to regulate cancer cells. For example, XIST sponges miR-200b-3p to promote ZEB1 expression to regulate metastasis and EMT in colorectal cancer [44]. XIST sponges miR-101 to promote EZH2 expression to affect gastric cancer progression [45]. XIST sponges miR-34a to stimulate MET protein expression and results in a promotion of tumor growth of thyroid cancer [41], etc. Similar molecular mechanism was identified in the present study. For the first time, our study demonstrated that XIST/miR-424/Shh axis is a novel regulatory pathway in regulating proliferation and activity of DP cells, as well as DP mediated hair follicle regeneration. Other lncRNAs and downstream target genes might also bind to these nucleotide sequences. In addition to miR-424, other miRNAs belonging to the miR-15–16 family might also participate in the regulation of this system [22]. Also, miR-424 may regulate or the potentail target genes to regulate DP cells, like AKT3 and PSAT1 [29], and XIST may target to other miRNAs, like miR-133a [39]. Wnt/β-catenin signaling pathway were involved in regulating hair follicle regeneration [30,38], and PlncRNA-1 promoted proliferation and differentiation of hair follicle stem cells through upregulation of TGF-β1mediated Wnt/β-catenin signaling pathway [30]. It was reported that XIST activated Wnt/β-catenin signaling pathway by sponging miR-34a in colon cancer [46]. However, whether XIST regulates Wnt/β-catenin signaling pathway through miR-424 in hair follicle regeneration is worth further investigation. Besides, according to previous reports [47], hedgehog signaling regulated miR-15–16 family to affect neural stem cell activity. Thus, it may be rational to propose that miR-424 may be regulated by hedgehog signaling, and forms a miR-424/hedgehog feedback loop,which needs to be further verified in the future.

Taken together, the present study investigated the role of XIST in the regulation of DP mediated hair follicle regeneration. Our results demonstrated that XIST/miR-424/Shh regulatory axis is a novel and important mechanism for facilitating DP activities, subsequently hair follicle regeneration and hair neogenesis. This new mechanism may provide new target for the development of therapeutic agents to stimulate hair neogenesis and the treatment of alopecia. Acknowledgements This work was supported by National Natural Science Foundation of China (Grant No.81701938), Guangxi Provincial Natural Science Foundation of China (Grant No.2018GXNSFBA050025) and China Postdoctoral Science Foundation (Grant No.2019M6). Declaration of Competing Interest None declared. References [1] A. Ho, J. Shapiro, Medical therapy for frontal fibrosing alopecia: a review and clinical approach, J. Am. Acad. Dermatol. 81 (2019) 568–580. [2] S. Qi, F. Xu, Y. Sheng, Q. Yang, Assessing quality of life in alopecia areata patients in China, Psychol. Health Med. 20 (2015) 97–102. [3] R.L. Girijala, R.R. Riahi, P.R. Cohen, Platelet-rich plasma for androgenic alopecia treatment: a comprehensive review, Dermatol. Online J. 24 (2018). [4] E.L. Crowley, S.C. Fine, K.K. Katipunan, M.J. Gooderham, The use of Janus kinase inhibitors in alopecia Areata: a review of the literature, J. Cutan. Med. Surg. 23 (2019) 289–297. [5] C. Ekelem, C. Pham, N. Atanaskova Mesinkovska, A systematic review of the outcome of hair transplantation in primary scarring alopecia, Skin Append. Disord. 5 (2019) 65–71.

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