Patients and clinical samples
A total 25 patients with deep partial thickness burn (n = 25) were recruited from Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology in this research. The samples were harvested from patients during tangential excision of eschar at the 5th day after thermal injure. The normal skin tissues (n = 25) were remnant donor skin from trunk. Approvals of this study were obtained by the Ethics Committee of Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology. All participants have signed the informed consent. All samples were stored at − 80 °C for following analyses.
Cell culture and treatment
Human skin fibroblast (HSF) cells were purchased from Gefanbio (Shanghai, China). The cells were cultured with RPMI-1640 medium (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (Gibco) in an incubator with 5% CO2 atmosphere at 37 °C.
To establish the cellular model of thermal injury, HSF cells were challenged in 52 °C water for 30 s, while cells in control group were incubated in 37 °C water for same time [7]. Subsequently, cells were cultured in the incubator at normal.
For cell transfection, small interfering RNA (siRNA) against XIST (si-XIST), COL1A1 (si-COL1A1), negative control (si-NC), pcDNA targeting XIST overexpression vector (XIST), COL1A1 overexpression vector (COL1A1), pcDNA empty vector, miR-29b-3p mimic (miR-29b-3p), miRNA negative control (miR-NC), miR-29b-3p inhibitor (in-miR-29b-3p) and inhibitor negative control (in-miR-NC) were synthesized by Genepharma (Shanghai, China). Cell transfection was performed in HSF cells using LipoFiter™ Liposomal Transfection Reagent (Hanbio, Shanghai, China) when cells reached 60–70% confluence in 6-well plates. After 24 h of the transfection, cells were harvested for thermal injury or following analyses.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using Trizol-based methods and the concentration and purity were analyzed through a NanoDrop ND-2000 spectrophotometer (Thermo Fisher, Wilmington, DE, USA). The RNA was reversely transcribed to cDNA using Taqman™ miRNA or mRNA Reverse Transcription Kit (Thermo Fisher). The qRT-PCR was performed with SYBR and special primers as follows: XIST (Forward, 5′-AATGACTGACCACTGCTGGG-3′; Reverse, 5′-GTGTAGGTGGTTCCCCAAGG-3′); COL1A1 (Forward, 5′-CCGTGCCCTGCCAGATC-3′; Reverse, 5′-CAGTTCTTGATTTCGTCGCAGATC-3′); β-actin (Forward, 5′-ATGGGTCAGAAGGATTCCTATGTG-3′; Reverse, 5′-CTTCATGAGGTAGTCAGTCAGGTC-3′); miR-29b-3p (Forward, 5′-TGCGGTAGCACCATTTGAAAT-3′; Reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′); U6 (Forward, 5′-TCCGATCGTGAAGCGTTC-3′; Reverse, 5′-GTGCAGGGTCCGAGGT-3′). The samples were prepared in duplicate. β-actin or U6 was regarded as internal reference for XIST, COL1A1 or miR-29b-3p, respectively. Their relative expression levels were analyzed according to the 2−ΔΔCt method [15].
Western blot
After washed with PBS, HSF cells were harvested for total protein extraction using RIPA lysis buffer (Yeasen, Shanghai, China). Following the high-speed centrifugation at 4 °C, the protein in supernatant was quantified according to the bicinchoninic acid (BCA) method with a BCA protein quantification kit (Yeasen). Equal amounts (20 μg) of proteins were prepared for SDS-PAGE electrophoresis and then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) by transmembrane with western transfer buffer (Beyotime, Shanghai, China). Each sample was prepared in triplicate. The membranes were blocked using QuickBlock™ Blocking Buffer (Beyotime), interacted with primary antibodies against Collagen I (ab34710, Abcam, Cambridge, MA, USA), alpha smooth muscle actin (α-SMA) (ab32575, Abcam), COL1A1 (sc-293182, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or β-actin (ab8227, Abcam) overnight at 4 °C, and incubated with special horseradish peroxidase-labeled secondary antibody (ab6721 or ab6728, Abcam) for 2 h. Protein blot was visualized in the dark using BeyoECL Plus (Beyotime) and films (Carestream Health, Rochester, NY, USA). The relative expression of protein was analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA) with β-actin as an endogenous control.
Cell proliferation
Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) (Beyotime). Transfected HSF cells were seeded into 96-well plates at a density of 3000 cells per well overnight and every sample was prepared in quadruplicate. At 0, 24, 48 or 72 h after thermal injury, cells were incubated with 10 μl CCK-8 solution for another 3 h. Subsequently, the absorbance at 450 nm was determined using a microplate reader (Bio-Rad).
Trans-well assay
The 24-well trans-well chambers (Corning, Corning, NY, USA) were used for investigation of migrated ability. Thermal injured cells in serum-free medium were seeded into upper chambers (1 × 104 cells) and the lower chambers were added with 500 μl medium containing fetal bovine serum. After a culture of 12 h at 37 °C, cells migrated to the lower surface were stained with 0.1% crystal violet (Sigma, St. Louis, MO, USA) and counted under an inverted microscope (Olympus, Tokyo, Japan) with three random fields.
Bioinformatics analysis and luciferase reporter assay
Bioinformatics analysis was used for explore the potential targets of XIST or miR-29b-3p by using DIANA tools and TargetScan. The putative binding sites of miR-29b-3p and XIST or COL1A1 were predicted. For luciferase reporter assay, wild type (WT) or mutant (MUT) luciferase reporter vectors targeting XIST or COL1A1 were established in firefly luciferase-expressing pmirGLO vector (Promega, Madison, WI, USA), named as XIST-WT, XIST-MUT, COL1A1-WT or COL1A1-MUT respectively. HSF cells were co-transfected with miR-29b-3p, miR-NC, in-miR-29b-3p or in-miR-NC and WT or MUT luciferase reporter constructs, along with renilla vector using LipoFiter™ Liposomal Transfection Reagent. At 48 h after post-transfection, luciferase activity was measured using a luciferase reporter assay kit (Promega).
Statistical analysis
All experiments were repeated three times. Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Inc., La Jolla, CA, USA) with the data expressed as mean ± standard deviation (S.D.). The comparisons between groups were conducted by student’s t test or ANOVA followed by Dunnett’s test. The spearman’s correlation analysis was performed to analyze the potential linear relationship of expression level of miR-29b-3p and XIST or COL1A1. The difference was significant when P < 0.05.