Expression and effect of microRNA-205 in hypertrophic scar

Abstract

Objective: To investigate the expression and effect of microRNA-205 (miR-205) in hypertrophic scar. Methods: The experimental research method were applied. From October 2019 to January 2020, hypertrophic scar tissue from 6 patients with hypertrophic scar [1 male and 5 females, aged (36±7) years], and remaining normal skin tissue from 6 trauma patients [2 males and 4 females, aged (38±9) years] after flap transplantation operation were collected. The above-mentioned 12 patients were admitted to the General Hospital of Northern Theater Command and met the inclusion criteria. Real time fluorescent quantitative polymerase chain reaction was used to detect the mRNA expressions of miR-205 and thrombospondin-1 (TSP-1). The hypertrophic scar tissue was taken to culture the 3rd to 5th passage of fibroblasts (Fbs) for the follow-up experiments. Fbs of hypertrophic scar was divided into TSP-1+miR-205 control group, TSP-1+miR-205 mimic group, TSP-1 mutant+miR-205 control group, TSP-1 mutant +miR-205 mimic group, which were transfected with the corresponding sequences. At 48 h after transfection, the expressions of luciferase and renal luciferase were detected by luciferase reporter gene detection kit, and the luciferase/renal luciferase ratio was calculated to indicate the activity of TSP-1. Two batches of hypertrophic scar Fbs were collected and divided into miR-205 control group, miR-205 mimic group, and miR-205 inhibitor group and miR-205 control group, miR-205 mimic group, and miR-205 mimic+TSP-1 group, respectively, which were transfected with the corresponding sequences. At 0 (immediately), 12, 24, 36, and 48 h after transfection, the cell viability was detected by microplate reader. Two batches of hypertrophic scar Fbs were collected, grouped, and treated as the cell viability detecting experiment. At 24 h after transfection, Hoechst 33258 staining was performed to observe the nuclear shrinkage, so as to reflect the apoptosis of Fbs. The number of samples in cell experiment was 3. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and t test. Results: The mRNA expression of miR-205 in hypertrophic scar tissue was 0.54±0.05, which was significantly lower than 1.26±0.07 in normal skin tissue (t=8.213, P<0.01). The expression of TSP-1 mRNA in hypertrophic scar tissue was 1.46±0.07, which was significantly higher than 0.68±0.11 in normal skin tissue (t=6.031, P<0.01). At 48 h after transfection, the luciferase/renal luciferase ratio reflecting the TSP-1 activity of cells in TSP-1+miR-205 mimic group was 0.532±0.028, which was significantly lower than 0.998±0.012 in TSP-1+miR-205 control group (t=26.500, P<0.01), and the luciferase/renal luciferase ratio of cells in TSP-1 mutant+miR-205 mimic group was 0.963±0.012, which was close to 0.976±0.010 in TSP-1 mutant+miR-205 control group (t=0.816, P>0.05). At 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 mimic group was significantly lower than that in miR-205 control group (t=6.169, 12.670, 27.130, 12.670, P<0.05 or P<0.01). At 0, 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 inhibitor group was significantly higher than that in miR-205 control group (t=6.169, 7.221, 7.787, 7.835, 13.030, P<0.05 or P<0.01). At 12, 24, 36, and 48 h after transfection, the cell viability in miR-205 mimic group was significantly lower than that in miR-205 control group and miR-205 mimic+TSP-1 group (t=8.118, 26.970, 39.550, 42.490, 14.570, 12.240, 36.830, 45.220, P<0.05 or P<0.01). At 24 h after transfection, compared with miR-205 control group, the cell apoptosis in miR-205 mimic group was increased, and the cell apoptosis in miR-205 inhibitor group was decreased. At 24 h after transfection, compared with miR-205 mimic group, the cell apoptosis in miR-205 control group miR-205 mimic+TSP-1 group were decreased. Conclusions: miR-205 can inhibit the proliferation and promote the apoptosis of Fbs in hypertrophic scar by inhibiting the expression of TSP-1, which has the potential to be the therapeutic target for hypertrophic scar.