Ethics statement
This study was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University and the informed consent of the patients was obtained. We performed this study following the principles recommended by Declaration of Helsinki. All experimental procedures involving animals were performed in accordance with animal protocols approved by the Institutional Animal Use and Care Committee.
Hair follicle stem cell isolation
Scalp tissues were obtained from 9 patients (6 males and 3 females) with a scalp laceration and contusion at the First Affiliated Hospital of Zhengzhou University. HFSc were isolated as described previously [15] with minor changes to the protocol. The scalp tissues were rinsed with penicillin-containing Hank's solution for 3 times and penicillin-free Hank's solution for 2 times. The scalp tissues were cut into 2mm × 2mm size and digested with 0.48U/mL neutral protease for overnight at 4 ℃. The intact hair follicles were gently extracted from hair follicles with surgical tweezers. Add 0.05% trypsin and 0.02% ethylene diamine tetraacetic acid (EDTA) mixture into the cut hair follicles tissue and digest at 37 ℃ for 30 min. Terminate the digestion then filter with 100 mesh steel mesh, and centrifuge the supernatant with 1000 rpm for 5 min. Added the Dulbecco’s modified eagle medium (DMEM) F12 (3: 1), insulin (5 mg/L), transferrin (5 mg/L), hydrocortisone (0.4 mg/L), EGF (20 μg/L), amphotericin B (2.5 mg/L), penicillin (1051 U/ L), streptomycin (100 mg/L) and 20% fetal bovine serum (FBS) into centrifugal. The cells were cultured at 5% CO2 and 37 ℃ saturated humidity. Finally, flow cytometry was used to detect the surface markers including CK14, CD200, Integrin α 6, p63 of HFSc to identify the successful isolation.
Differentiation and transfection of HFSc
HFSc were differentiated into adipocytes according to previously described methods [16]. Briefly, primary HFSc were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin (growth medium) in a 5% CO2 humidified atmosphere at 37°C. Forty-eight hours after confluence, differentiation was induced with DMEM supplemented with 10% FBS, 1 μM dexamethasone, 10 μg/mL insulin, 0.2 mmol/L indomethacin, 0.1 mmol/L ascorbic acid and 0.5 mM 3-isobutyl-1-methylxan-thine (IBMX) (MDI medium). Two days later, the medium was replaced with DMEM (10% FBS) and was changed every 2 d.
To construct lentiviral vectors overexpressing DNMT1 and MAPK1 and miR-214-3p gene, the human DNMT1 and MAPK1 and miR-214-3p gene DNA fragment was PCR amplified from human SGC7901 cell genomic DNA. The PCR-amplified fragments were inserted into a lentiviral vector pLV-EF1α-MCSIRES-Puro (pLV-ctrl) to generate pLV-DNMT1 and pLV-MAPK1 and pLV- miR-214-3p. Viral vector pLV-DNMT1 and pLV-MAPK1 and pLV- miR-214-3p and pLV-ctrl were transfected into HEK293T cells. Media containing lentiviruses (pLV-DNMT1 and pLV-MAPK1 and pLV- miR-214-3p and pLV-ctrl) were collected every 24 h for three times and the lentiviruses were purified by ultra-speed centrifugation. Full-length cDNA encoding human DNMT1 and MAPK1 and miR-214-3p were amplified by PCR, and the PCR product was sub-cloned into pBOBI and pCMV-HA vectors to obtain DNMT1 and MAPK1 and miR-214-3p gene overexpressing plasmids.
For cell transient transfection, the HFSc were seeded and grown in 6-well plates overnight. The short interfering RNA (siRNA) was used for inhibiting endogenous RNA expression [17]. The HFSc were transfected with negative control siRNA oligonucleotides, positive control siRNA oligonucleotides, siRNA against DNMT1 and miR-214-3p, using by Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Besides, the HFSc were transfected with DNMT1 and MAPK1 and miR-214-3p gene overexpressing plasmids. The PD98059 (19-143, Sigma, St. Louis, MO, USA), a MAPK1 inhibitor, was also administrated in HFSc. Subsequent experiments were carried out 48 h after transfection.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
TRIzol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from cells, and the total RNA concentration and purity were detected by nanodrop2000 microultraviolet spectrophotometer (1011U, Nanodrop, USA). The RNA was reversely-transcribed to complementary (cDNA) according to the instructions of TaqMan MicroRNA Assays Reverse Transcription primer (4427975, Applied Biosystems, USA), and the primer of miR-214-3p was designed and synthesized by TaKaRa (Table 1) with GAPDH as a control. For miRNAs, qPCR was performed with the stem-loop primers as reported previously [18]. U6 RNA served as an internal control. Each experiment was repeated three times.
Western blotting
Total protein was extracted from radio immunoprecipitation assay (RIPA) lysis buffer containing phenylmethanesulfonyl fluoride (PMSF) (P0013C, Beyotime Biotechnology, Shanghai, China) on ice for 30 min, centrifuged at 8,000g for 10 min at 4 ℃. The 50 ug protein was dissolved in 2 × SDS loading buffer and boiled at 100℃ for 5 min. Proteins were separated by electrophoresis on 8% to 12% SDS-polyacrylamide gels and transferred moist to polyvinylidene difluoride membranes. Membranes were blocked by 5% nonfat dry milk in PBS and incubated with antibodies for DNMT1 (1:1000, ab188453, Abcam, Cambridge, UK), MAPK1 (1:200, R&D, MAB1230-SP), ERK1/2 (1:10000, ab184699, Abcam), phosphorylated (p)-ERK1/2 (1:1000, ab214362, Abcam), PPAR-γ2 (1:500, ab23673, Abcam), perilipin (1:1500, ab3526, Abcam), Adipoq (1:1000, ab62551, Abcam), aP2 (1:1000, ab218107, Abcam) and GAPDH (1:2500, ab9485, Abcam) overnight at 4°C. The membrane was incubated with HRP-labeled goat anti-rabbit IgG (1:2000, ab97051, Abcam) for 1 h. The ECL fluorescence detection kit (BB-3501, Ameshame, UK) was added on the membrane, then using Bio-Rad image analysis system (Bio-Rad, USA) with Quantity One v4.6.2 analysis software to analysis and GAPDH was used as internal control. The experiment was repeated three times. Oil-red-O staining
The HFSc were cultured in DMEM/F12 medium and collected on 7d and 14d, respectively. The cells were fixed with 10% formalin, washed with 60% isopropanol, and stained with oil red O working fluid. Being fixed by glycerine gelatin, cells were observed under a microscope (Olympus optics, Tokyo, Japan). The number of positive cells stained with oil red O was counted under the microscope.
Dual-luciferase reporter assay
MAPK1 was identified as a miR-214-3p target in TargetScan7.1 (http://www.targetscan.org/vert_71/). Human HEK293T cells were cultured in DMEM medium containing 10% FBS. The cDNA fragment of MAPK1 3' -untranslated region (UTR), MAPK1-wild type (Wt) containing the miR-214-3p binding site was inserted into the pmiRGLO vector. The cDNA fragment of MAPK1 3' -UTR, MAPK1-mutant type (Mut) was synthesized by point mutation and inserted into pmiRGLO vector. The inserted sequence was verified to be correct by sequencing performed by RiboBio Co., LTD (Shanghai, China). The recombinant vector pmiRGLO-MAPK1-Wt or pmiRGLO-MAPK1-Mut was co-transfected with a miR-214-3p mimic (miR-214-3p overexpression sequence) or an NC mimic (negative control sequence) into HEK293T cells by liposome transfection, and the cells were incubated and cultured for 48 h before being collected and lysed. Take 100 μL lysate supernatant of was taken and 100 μL Renilla luciferase assay solution was added to detect the activity. In addition, take 100 μL lysate supernatant and 100 μL firefly luciferase to detect the luciferase activity. After 48 hours, the cells were collected, and the luciferase and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity. The SpectraMax M5 (Molecular devices instruments CO., LTD., Shanghai, China, Origin: USA) with an interval of 2 s and a determination time design of 10 s was used to detect the luciferase activity of Renilla luciferase and Firefly luciferase, respectively.
Real-time quantitative methylation-specific PCR (MSP)
The methylation of gene promoter was detected by MSP. Genomic DNA was extracted by Genomic DNA extraction kit (Tiangen Biochemical technology CO., LTD, Beijing, China) according to the instructions. The DNA concentration and purity were determined by ultraviolet spectrophotometry. DNA was treated with sodium sulfite using the EZ DNA Methylation Kit (Zymo Research, USA), and the reaction column was used for desulfurization and purification. The purified DNA could be used for subsequent PCR reaction. The methylation and non-methylation primers (Table 2) were designed for the miR-214-3p gene promoter by CpG island enrichment area. The reaction products were subjected to agarose gel electrophoresis, gel electrophoresis imaging and image analysis system. If the CpG island in the promoter region is completely methylated, only the methylated primer can amplify the target band. If there is no methylation, only non-methylated primers can amplify the target band. If partial methylation occurs, the target bands can be amplified from both primers. Partial methylation is classified as methylation. Serial dilutions of plasmid DNA was used as standards for quantification [19]. Each experiment was repeated three times.
Chromatin immunoprecipitation (ChIP)
Differentiated and cultured HFSc were taken, the cell fusion degree reached 70-80%, 1% formaldehyde was added and fixed at room temperature for 10 min to make the DNA and protein in the cell fixed and cross-linked. Then, the protein was broken randomly by ultrasonic treatment for 10 s, and interval for 10 s and 15 cycles and to make fragments of appropriate size. The supernatant was collected by centrifugation at 13000 rpm and divided into two tubes, and added with antibody for negative control and rabbit anti-IgG (1: 100, ab172730, Abcam, UK) and mouse anti-DNMT1 (1: 100, ab13537, Abcam, UK) at 4℃ for overnight, respectively. The endogenous DNA-Protein complex was precipitated by Protein Agarose/Sepharose, and the non-specific complex was washed, the cross-linking was performed overnight at 65℃, and the DNA fragments were extracted and purified by phenol and chloroform to detect the binding of miR-214-3p gene promoter fragment to DNMT1.
Mouse model of trauma
A total of 24 mice (weight 20 ± 2 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. The mice were anesthetized by intraperitoneal injection with 3% barbiturate. Two wounds at a position of 1.0 cm on both sides of the back spinal column were created to form a circular skin incision and not touch the muscles. After the wound was formed, it was not bandaged and treated with medicine. The mice was kept separately in a sterile laboratory and sterilized every day. Lentivirus vectors (5× 108 pfu/100 L) were introduced into the wound surface of mice beside the wound surface. The mice were randomly divided into oe-NC+sh-NC group (n=8), and oe-DNMT1+sh-NC group (n=8) and oe-DNMT1+sh-MAPK1 group (n=8). The overexpressing lentivirus vectors were used by LV5-GFP and silencing lentivirus vectors were used by pSIH1-H1-copGFP. The wound area of each group was photographed and recorded on day 0, 6, 10 and 14, respectively. The mice were sacrificed on day 18, and skin tissue was extracted from the wound. The tissue sections were embedded by paraffin or used to extract proteins for detection.
Immunofluorescence staining
Mouse skin tissues were fixed with 4% paraformaldehyde for overnight. Then, the tissue was washed and sealed with normal saline of 0.01 M phosphate buffer for three times, and then sealed with 10% goat serum at room temperature for 30 min. After that, TGF-β1 (1:200, ab92486, abcam, Cambridge, UK), vascular endothelial growth factor (VEGF) (1:200, ab2350, abcam, Cambridge, UK) and platelet derived growth factor (PDGF-BB) (1:200, ab9704, abcam, Cambridge, UK) were incubated at 4℃ for overnight. The second antibody and DAPI were incubated at room temperature in the dark for 1 hour, and glycerol was fixed. The confocal laser scanning microscope was used to analyze (LSM, FV1000; Olympus corp., Tokyo, Japan).
Statistical analysis
All statistical tests were performed using SPSS 21.0 (IBM SPSS Statistics, Armonk, NY, USA). The data were presented as mean ± standard deviation. The data in the two groups were compared by unpaired Student's t test, and the data in multiple groups were compared by one-way analysis of variance (ANOVA) and Tukey's post-test. p < 0.05 was considered statistically significant.