Cell culture and treatment
All HTSs were obtained from patients undergoing HTS resection surgery at the Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. The study protocol was approved by the ethics committee of our institute (ethics number: SH9H-2020-A126-1). A total of 6 subjects with HTSs were enrolled in this study, and the characteristics of these patients are shown in Table 1. HTS tissues were minced, immersed in 0.25% Dispase II (Roche, Switzerland), and incubated at 37 °C for 1 hour. The epidermis was then separated and digested with 0.2% NB4 (Roche, Switzerland) at 37 °C for 2-3 hours to isolate the HSFs. The collected HSFs were cultured in complete high-glucose Dulbecco’s modified Eagle’s medium (CM) (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 100 U/ml penicillin and streptomycin (Gibco, USA) and incubated at 37 °C in 5% CO2. The medium was changed every 3 days, and all the experiments were performed with cells from the third to sixth passages (P3-P6).
The siRNA oligonucleotides and HKP (a biodegradable polypeptide cationic molecule) were provided by Sirnaomics (Suzhou, China). HSFs were seeded in the wells of 6-well plates at a density of 106 cells in 3 ml media per well. Six hours later, the media was replaced with fresh media supplemented with HKP alone, HKP (NC siRNA), HKP (COX-2/TGF-β1 siRNAs), HKP (TGF-β1 siRNA), or HKP (COX-2 siRNA) at concentrations of 4 ug/ml HKP loaded with 1 ug/ml siRNA and incubated for 48 hours unless otherwise stated.
Table 1. The characteristics of each sample.
Patient number
|
Sex
|
Age
|
Biopsy site
|
Treatment before surgery
|
1
|
Male
|
25
|
Arm
|
None
|
2
|
Male
|
54
|
Neck
|
None
|
3
|
Male
|
35
|
Chest
|
None
|
4
|
Female
|
26
|
Abdomen
|
None
|
5
|
Male
|
15
|
Arm
|
None
|
6
|
Male
|
64
|
Abdomen
|
None
|
Cell Counting Kit-8 (CCK-8) assay
The proliferation of HSFs was measured by CCK-8 assay. HSFs were seeded into the wells of 96-well plates at a density of 3×104 cells in 100 µl media per well. After adherence, the media were replaced with fresh media supplemented with HKP alone, HKP (NC siRNA), HKP (0.5, 1, 2, 4, 8 µg/ml TGF-β1 siRNA), HKP (0.5, 1, 2, 4, 8 µg/ml COX-2 siRNA) or HKP (1, 2, 4, 8, 16 µg/ml COX-2/TGF-β1 siRNAs). Five replicates were established for each concentration. After treatment for 48 hours, 10 µl CCK-8 solution (Dojindo, Japan) was added to each well and incubated for 2 hours at 37 °C. Cell viability was quantified by measuring the absorbance at 450 nm with a microplate reader (Synergy H (1), Biotek, Vermont, USA). Three biological replicates were performed for the CCK-8 assay.
Quantitative real-time PCR (qRT–PCR)
After HSFs were transfected, total RNA was extracted by TRIzol Reagent (Invitrogen, USA). A spectrophotometer (NanoDrop2000, USA) was then used to assess the purity of the RNA. cDNA was synthesized by the reverse transcription of 1000 ng RNA according to the manufacturer’s protocols (Takara, Japan). Gene expression levels of COX-2, TGF-β1, Col I, Col III, α-SMA, TP53, Bcl-2, Bax, Caspase-3 and Caspase-9 were measured by qRT–PCR using the TB GREEN Premix Ex Taq™ kit (Takara, Japan), and gene expression was normalized to β-actin expression on an ABI QuantStudio6 Pro (Applied Biosystems, USA). The primer sequences used in this study are presented in Table 2. Five biological replicates were performed for the qRT–PCR.
Table 2. Primer sequences used in the study for qRT–PCR analyses.
Gene
|
Forward sequence
|
Reverse sequence
|
hβ-actin
|
5′-ACCAACTGGGACGACATGGAGAAA-3′
|
5′-TAGCACAGCCTGGATAGCAACGTA-3′
|
hTGFβ1
|
5′-GAGCCTGAGGCCGACTACTA-3′
|
5′-CGGAGCTCTGATGTGTTGAA-3′
|
hCOX-2
|
5′-ATTCCCTTCCTTCGAAATGC-3′
|
5′-GGGGATCAGGGATGAACTTT-3′
|
hα-SMA
|
5′-GGCATTCACGAGACCACCTAC-3′
|
5′-GGGGCGATGATCTTGATCTT-3′
|
hCOL1A1
|
5′-AGGGCCAAGACGAAGACATC-3′
|
5′-GTCGGTGGGTGACTCTGAGC-3′
|
hCOL3A1
|
5′-TGAAGGGCAGGGAACAACT-3′
|
5′-GGATGAAGCAGAGCGAGAAG-3′
|
hCaspase-3
|
5′-CCAAAGATCATACATGGAAGCG-3′
|
5′-CTGAATGTTTCCCTGAGGTTTG-3′
|
hTP53
|
5′-TTCCTGAAAACAACGTTCTGTC-3′
|
5′-AACCATTGTTCAATATCGTCCG-3′
|
hBCL-2
|
5′-GACTTCGCCGAGATGTCCAG-3′
|
5′-GAACTCAAAGAAGGCCACAATC-3′
|
hBAX
|
5′-CGAACTGGACAGTAACATGGAG-3′
|
5′-CAGTTTGCTGGCAAAGTAGAAA-3′
|
rβ-actin
|
5′-AGCCACCAATCCACACAG-3′
|
5′-CCTCTATGCCAACACAGT-3′
|
rTGFβ1
|
5′-CTAATGGTGGACCGCAACAAC-3′
|
5′-CACTGCTTCCCGAATGTCTGA-3′
|
rCOX-2
|
5′-AACACCTGAGCGGTTACCACT-3′
|
5′-GAGGCAATGCGGTTCTGATAC-3′
|
rTP53
|
5′-GTCAGGGACAGCCAAGTCT-3′
|
5′-CGTCTCACGACCTCAGTCAT-3′
|
rCaspase-3
|
5′-ATAAAAATGACCTTACTCGTGAA-3′
|
5′-AGTAGTCGCCTCTGAAGAAAC-3′
|
rCaspase-9
|
5′-GGACTGTCTTTTGCCTGTATC-3′
|
5′-GACCTTGGAACACAGAGAAATAC-3′
|
Western blotting analysis
The HSFs were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1 mM phenylmethyl sulfonyl fluoride (PMSF) for 30 min on ice and then centrifuged. The protein concentrations of the supernatants were measured with a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). The extracted proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to 0.45 µm polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After blocking with 5% bovine serum albumin (BSA) for 1 hour at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies were anti-β-actin (ab16039; Abcam, UK), anti-type I collagen (Col I, ab34710, Abcam, UK), anti-type Ⅲ collagen (Col Ⅲ, ab7778, Abcam, UK), anti-α-SMA (#48938, Cell signaling technology, USA), anti-TGF-β1 (#84912, Cell signaling technology, USA), anti-COX-2 (#4842, Cell signaling technology, USA), anti-TP53 (#2527, Cell signaling technology, USA), anti-Bcl-2 (#4223, Cell signaling technology, USA), anti-BAX (#5023, Cell signaling technology, USA), anti-Caspase-3 (#9662; Cell Signaling Technology, USA), anti-cleaved Caspase-3 (#9664; Cell Signaling Technology, USA), anti-phosphor-MEK (AF3385, Affinity, USA), anti-phosphor-ERK (AF1015, Affinity, USA), anti-AKT (#4060; Cell Signaling Technology, USA) and anti-PI3K(#4255; Cell Signaling Technology, USA) antibodies. Horseradish peroxidase (HRP)-conjugated secondary antibodies (#7074, Cell Signaling Technology, USA) were added on the second day and incubated at room temperature for 1 hour. The protein expression levels were analyzed with Odyssey V3.0 image scanning (Li-COR. Inc., Lincoln, NE, USA). The reference gene β-actin (Abcam, UK) was used as a loading control. Western blot assays were conducted on three biological replicates.
Wound healing assay
Briefly, HSFs were seeded in 6-well plates at a density of 6 × 104 cells/cm2 and cultured with CM until confluence. A scratch wound was then made in the middle of each well with 1 ml pipette tips. Floating cells were removed by washing with phosphate-buffered saline (PBS) three times. The HSFs in each well were transfected with the corresponding siRNAs, and the width of the scratch was recorded in five random fields at 0, 12, 24 and 36 hours. Five biological replicates were performed for the wound healing assay.
Transwell assay
HSFs were added to the upper chambers of Millicell Hanging Cell Culture Inserts with 8-μm pores (Millipore, USA) at a density of 1×105 cells/ml in 200 μl serum-free DMEM, and 500 μl of DMEM supplemented with 10% FBS and the indicated siRNAs was added to the lower chambers. After 24 hours of incubation, the chambers were removed and fixed with 4% paraformaldehyde (PFA) for 5 min, gently washed and stained with crystal violet for 10 min. The migrated cells that had entered the lower chamber were quantified in each field of images captured under a microscope (Nikon, Japan). The transwell assay was performed on three biological replicates.
EdU assay
HSFs were seeded in the wells of 96-well plates at a density of 3×104 cells in 100 µl media per well. EdU (RiboBio, China) was dissolved in CM at a concentration of 50 μM, and 100 µl was added to each well. After incubation for 2 hours and washed, the cells were fixed in 4% PFA for 30 min. The cells were incubated with 100 μl 0.5% Triton X-100 (Sigma, USA) for 10 min and washed twice. For the EdU click reaction, 100 μl of RiboBio's staining solution was added, and incubated for 30 min in the dark then stained the nuclei with Hoechst (Invitrogen, USA). A total of 5 images were randomly selected and high-magnification views were used to calculate the percentage of positive cells with ImageJ software (National Institutes of Health, USA). The data were analyzed based on five biological replicates.
Immunofluorescence analysis
The transfected HSFs were fixed with 4% PFA and permeabilized with 0.5% Triton X-100 (Sigma, USA) for 20 min. After blocking with 5% goat serum (Gibco, USA) for 1 hour at room temperature, the slides were incubated with a rabbit monoclonal anti-α-SMA antibody (ab5694, Abcam, UK) at 4 °C overnight. The slides were incubated with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (ab150077, Abcam, UK) for 1 hour nextday. Finally, the slides were sealed with DAPI Fluoromount-G, and the fluorescence was analyzed by confocal microscopy (Nikon, Japan). Immunofluorescence analysis was conducted on at least three biological replicates.
Annexin V/PI staining
The transfected cells were digested with 0.25% trypsin (Gibco, USA) and washed with cold PBS. Cell apoptosis was assessed using a FITC Annexin V Apoptosis Detection Kit (BD, USA) according to the experimental instructions. The stained cells were analyzed by flow cytometry (Beckman, USA), and the data were analyzed by FlowJo software (Treestar, USA) based on at least three biological replicates.
Animal model
For the animal model, HTSs were generated in the tails of twelve 8-week-old male Sprague–Dawley rats as we previously described35. Briefly, 6 × 6-mm wounds were created on the tails. After allowing re-epithelialization to occur for approximately 2 weeks, the wounds were stretched with continuous strain to induce the formation of HTSs for another two weeks (Supplementary Figure 2A-B). Twelve weeks after being subjected to mechanical strain, the scar formed on the rat tails and the rats were randomly divided into four groups: the HKP (NC siRNA), HKP (COX-2 siRNA), HKP (TGF-β1 siRNA), and HKP (COX-2/TGF-β1 siRNAs) groups. HKP (4 µg/ml) loaded with the indicated siRNA (1 µg/ml) was injected into the scar every five days for 15 days. Scar samples were harvested after treatment for further evaluation. We used the scar elevation index (SEI), which has been reported in many studies on HS, to quantify scar formation29,30,41. The SEI ratio was calculated as follows: SEI = the length of the highest point of the scar to the surface / the length of the highest point of the adjacent normal skin to the surface of the tail. An SEI >1 indicates HS formation. The data was analyzed based on at least three biological replicates.
Histological staining
After fixing the harvested samples with 4% PFA for 24 hours, the tissues were paraffin-embedded and cut into 5-μm-thick sections. Tissue sections were stained with hematoxylin & eosin (H&E, Sigma–Aldrich) and observed by microscopy (Nikon, Japan). ImageJ software (National Institutes of Health, USA) was then used to measure and analyze the gross area of the generated scar. Cell apoptosis of the tail HTS samples was assessed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using the In Situ Cell Death Detection Kit (Roche, Switzerland) following the manufacturer’s instructions. Briefly, the sections were deparaffinized, blocked, and then stained with TUNEL test solution. After washing twice with PBS, the sections were incubated with an HRP-conjugated goat anti-rabbit secondary antibody at room temperature. The percent of TUNEL-positive cells was quantified by ImageJ software (National Institutes of Health, USA) based on six biological replicates.
Statistical analysis
The results are presented as the mean ± standard deviation (SD). The data between two groups were analyzed by Student’s t test to determine significance. For multivariate comparisons, one-way ANOVA with Tukey’s multiple comparison test was performed. P < 0.05 was considered statistically significant. Statistical analysis was performed with GraphPad Prism 9.0 software (GraphPad Software, USA).