HFTs were purchased from Beijing Jing-Meng High-Tech Stem Cell Technology Co., Ltd. (Beijing, China). They were cultured in Minimum Essential Medium (MEM) (Sigma Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂.

LncRNA5322 (19) or miR-21 (5′-UCAACAUCAGUCAGAUAAGCUA-3′) and negative control lncRNA-vector (18) or miR-vector (control, 5′-UAGCUUAUCAGACAGAUGUUGA-3′) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, USA). plncRNA5322 or pmiR-21 was cloned into the pBabe vector (Cell Biolabs, Inc., San Diego, CA, USA) to generate the plncRNA5322 and pmiR-21 vectors. Transfection of plncRNA5322, pmiR-21 and negative control vectors was performed using X-treme GENE RNA transfection reagent (Roche Applied Science, Rotkreuz, Switzerland). Transfection concentrations were 100 nM for plncRNA5322 and pmiR-21 or negative vector. After 48-h following transfection, cells were used to further analysis.

HFTs were cultured and treated with agomir-21 or PI3K inhibitor (0.5 mg/ml, Guangzhou RiboBio Co., Ltd., Guangzhou, China) at 37°C in a humidified atmosphere containing 5% CO₂. Cell proliferation and differentiation were analyzed as previously described (20,21). For cell differentiation, HFTs were cultured in MEM for 12 h at 37°C. HFT colonies growing on Matrigel (Corning China, Ltd., Shanghai, China) were loosely detached by dispase treatment for 5 min and washed 3 times with PBS. Cells were resuspended in Dulbecco's modified Eagle's medium (Sigma Aldrich; Merck KGaA) containing 20% FBS. Cells were maintained on 1% agar-coated slides and allowed to differentiate for another 18 days at 37°C. Cells were subsequently fixed with 10% formalin for 1 h at 37°C and stained with 60% Oil Red O in isopropanol as working solution for 10 min at 37°C. The proportion of Oil Red O-positive cells was determined by counting stained cells under a light microscope at 40× magnification. For cell proliferation assay, HFTs were seeded in 96-well plates (10³ cells/well) and cultured for 24 h at 37°C. Cells proliferation was determined using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol.

Total RNA was extracted from HFTs using the RNAeasy Mini kit (Qiagen Sciences, Inc., Gaithersburg, MD, USA) and 1 µg total RNA was transcribed into cDNA using an RT kit (Qiagen Sciences, Inc.) for 1.5 h at 42°C. The cDNA (10 ng) was subjected to a qPCR using a SYBR-Green Master Mix system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) and sequences are as follows: Cyclin-dependent kinase (CDK)1 forward, 5′-CAGACTAGAAAGTGAAGAGGAAGG-3′ and reverse, 5′-ACTGACCAGGAGGGATAGAATC-3′; CDK2 forward, 5′-TTGGCAGCACACTCTATG-3′ and reverse, 5′-CCTCATTCGGCAAATAAACG-3′; LncRNA5322 forward, 5′-GACGAACTGACCGGTTGTCT-3′ and reverse, 5′-GTGACAGAGGGATAGCGAGC-3′; and β-actin forward, 5′-CGGAGTCAACGGATTTGGTC-3′ and reverse, 5′-AGCCTTCTCCATGGTCGTGA-3′. The following thermocycling conditions were applied: 45 amplification cycles consisting of denaturation at 95°C for 30 sec, primer annealing at 63°C for 45 sec with touchdown to 57°C for 50 sec, and applicant extension at 72°C for 60 sec. Relative mRNA expression changes were calculated using the 2^−ΔΔCq method (22). The results are expressed as the n-fold way compared to control.

HFTs were collected and lysed in radioimmunoprecipitation assay buffer (mammalian protein extraction reagent for the cells and tissue protein extraction reagent for the tissues; Thermo Fisher Scientific, Inc.) followed by homogenization at 4°C for 10 min. Protein concentration was measured by a Biconchoninic Acid protein assay kit (Thermo Fisher Scientific, Inc). A total of 20 µg protein/lane was separated by 12.5% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated in blocking buffer (5% milk) for 2 h at 37°C prior to incubation with primary antibodies at 4°C overnight. The primary rabbit anti-mouse antibodies used in the present study were: CDK1 (ab133327; 1:1,000; Abcam, Cambridge, UK), CDK2 (ab76146; 1:1,000; Abcam), PI3K (ab182651; 1:200; Abcam), AKT (ab8805; 1:1,000; Abcam), phosphorylated (p)PI3K (ab182651; 1:1,000; Abcam), pAKT (ab38449; 1:500; Abcam) and β-actin (ab8226; 1:500; Abcam). A horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000; Bio-Rad Laboratories, Inc.) was used as the secondary antibody. Bands were visualized using Western Blotting Luminol Reagent (Pierce^™ Fast Western Blot kits, SuperSignal^™ West Femto; Thermo Fisher Scientific, Inc.). Bands intensities normalized to β-actin. The density of the bands was analyzed by Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.).

The 3′-untranslated region (3′-UTR) sequence of PI3K and AKT, predicted to interact with lncRNA AK015322 or lncRNA vector (control) sequence within the predicted target sites (http://www.cbcb.umd.edu/software/GeneSplicer/gene_spl.shtml), was inserted into the pGL3 control vector (Promega Corporation, Madison, WI, USA). These constructs were designated as PI3K-3′-UTR and AKT-3′-UTR, respectively. For the reporter assay, HFTs cells were seeded in 24-well plates and transfected with the above constructs using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.), lncRNA AK015322 expression vector and negative control. After 48 h, the cells were collected and Renilla luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer's protocols. Results were obtained from 3 independent experiments performed in duplicate.

Data are presented as the mean ± standard deviation of triplicate dependent experiments and analyzed using Student t-tests or one-way analysis of variance followed by Tukey's honest significant difference test. Significance was established using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The effect of lncRNA5322 on the proliferation and differentiation of HFTs was investigated. Transfection with lncRNA5322 increased lncRNA5322 expression in HFTs compared with the control (Fig. 1A). The results demonstrated that lncRNA5322 transfection stimulated proliferation of HFTs compared with control cells (Fig. 1B). The results also revealed that PlncRNA-1 transfection markedly promoted HFT differentiation (Fig. 1C). These results indicate that lncRNA5322 transfection induces the proliferation and differentiation of HFTs.

miR-21 transfection has been reported to promote the differentiation of hair follicle-derived neural crest stem cells into Schwann cells (23). In the present study, it was demonstrated that miR-21 expression levels were upregulated by lncRNA5322 transfection-induced differentiation (Fig. 2A). The results revealed that lncRNA5322 transfection promotes CDK1 and CDK2 mRNA and protein expression in HFTs (Fig. 2B-D). These results suggest that LncRNA5322 upregulates miR-21 expression and increases CDK1 and CDK2 expression during HFT differentiation.

The role of miR-21 in the proliferation and differentiation of HFTs was investigated. Results demonstrated that miR-21 transfection (pmiR-21; Fig. 3A) promoted HFT proliferation and differentiation (Fig. 3B and C). Transfection with miR-21 also upregulated CDK1 and CDK2 mRNA and protein expression levels during HFTs differentiation (Fig. 3D and E). Agomir-21 transfection blocked lncRNA5322-mediated HFT proliferation and differentiation (Fig. 3F and G). These results indicate that transfection with miR-21 promotes HFT proliferation and differentiation and increases cyclin expression during HFT differentiation.

A previous study indicated that the PI3K/AKT signaling pathway is associated with stem cell differentiation (24). The association between lncRNA5322 and the PI3K/AKT signaling pathway during HFT differentiation was analyzed. The results revealed that lncRNA5322 transfection increased PI3K and AKT expression and phosphorylation levels in HFTs (Fig. 4A and B). It was also demonstrated that PI3K inhibitor (PI3KIR) inhibited lncRNA5322-induced proliferation and differentiation of HFTs (Fig. 4C and D). Notably, the results of a luciferase gene report assay demonstrated that lncRNA5322 transfection increased the luciferase activity of PI3K and AKT (Fig. 4E). These results suggest that lncRNA5322 regulates proliferation and differentiation in HFTs via the PI3K/AKT signal pathway.

The effects of agomir-21 on the PI3K/AKT signaling pathway in HFTs were investigated. It was demonstrated that agomir-21 transfection decreased PI3K and AKT expression and phosphorylation levels in HFTs (Fig. 5A and B). Agomir-21 transfection also blocked the lncRNA5322-induced upregulation of the PI3K/AKT signaling pathway during HFT differentiation (Fig. 5C and D).