Proliferation of hypertrophic scar fibroblasts inhibited by microRNA-627 targeting IGF-Ⅰin hypertrophic scar

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Objective: To investigate the mechanism of microRNA-627(miR-627) inhibiting the proliferation of hypertrophic scar fibroblasts (Fbs) by targeting IGF-I. Methods: The experimental method was used. From October 2019 to January 2020, hypertrophic scar tissues from 6 patients with hypertrophic scar (2 males and 4 females, aged (34±11) years) and the remaining normal skin tissues from 6 patients with trauma (3 males and 3 females, aged (35±13) years) after skin flap transplantation were collected. the above-mentioned 12 patients were admitted to the General Hospital of Northern Theater Command and met inclusion criteria. The mRNA expression of miR-627 was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The 3rd to 5th generations of Fbs were cultured from hypertrophic scar tissue for subsequent experiments. Fbs from hypertrophic scar were divided into miR-627 control group, miR-627 mimic group and miR-627 inhibitor group. The corresponding sequences were transfected respectively. At 0 (immediate), 12, 24, 36 and 48 h after transfection, the cell viability was detected by thiazolyl blue reagent; at 24 h after transfection, the apoptosis was detected by Annexin V-fluorescein-5-isothiocyanate/propidium iodide kit; at 24 h after transfection, the expression levels of IGF-Ⅰ, collagen I and a-SMA were detected by Western blot. The hypertrophic scar Fbs were divided into IGF-Ⅰ wild type + miR-627 control group, IGF- wild type + miR-627 mimics group, IGF-Ⅰ mutant + miR-627 control group. At 48 hours after transfection, the expression of luciferase and renal luciferase were detected by luciferase reporter gene detection kit, and the ratio of the two was calculated to reflect the activity of IGF-Ⅰ. Fbs from hypertrophic scar were divided into miR-627 control group, miR-627 mimic group and miR-627 mimic + IGF-I group, and were transfected with corresponding sequences respectively. At 24 h after transfection, the expression levels of IGF-Ⅰ, type I collagen and a-SMA were detected by Western blot. The number of samples in cell experiment was 3. Analysis of variance, one-way analysis of variance, t test and chi-square test were used to statistic the data. Results: The expression of miR-627 mRNA in hypertrophic scar tissue was 0.47±0.06, which was significantly lower than that in normal tissue 1.12±0.23 (t=15.090, P<0.01). At 12, 24, 36 and 48 hours after transfection, the cell viability of miR-627 mimic group was significantly lower than that of miR-627 control group (t=9.918, 34.370, 13.580, 61.550, P<0.05 or P<0.01); the cell viability of miR-627 inhibitor group was significantly higher than that of miR-627 control group (t=4.722, 8.616, 13.330, 14.000, P<0.05 or P<0.01). At 24 h after transfection, the apoptosis rate of miR-627 mimic group was (10.89±0.35)% significantly higher than that of miR-627 control group (8.42±0.47)% (t=7.301, P<0.01), and that of miR-627 inhibitor group was (5.00±0.22)% significantly lower significantly (t=11.510, P<0.01). At 24 h after transfection, compared with miR-627 control group, miR-627 mimics could significantly down regulate the expression of IGF-Ⅰ, type I collagen and a-SMA (t=25.470, 5.282, 7.415, P<0.05); miR-627 inhibitor could up regulate the expression of IGF-Ⅰ, type I collagen and a-SMA (t=15.930, 8.857, 9.763, P<0.05). At 48 h after transfection, the luciferase/renal luciferase ratio of IGF-Ⅰ in IGF-Ⅰ wild type + miR-627 mimic group was 0.463±0.061, which was significantly lower than that of IGF-Ⅰ wild type + miR-627 control group 0.999±0.011 (t=16.852, P<0.01), The luciferase/renal luciferase ratio of IGF-mutant + miR-627 mimic group was 0.934±0.021, which was similar to that of IGF-Ⅰ mutant+miR-627 control group 0.930±0.023 (t=1.959, P>0.05). After 24 hours of transfection, the protein expressions of IGF-Ⅰ, collagen I and a-SMA in miR-627 mimic group were 1.623±0.070, 1.363±0.042 and 1.617±0.025, which were significantly lower than those in miR-627 control group 2.723±0.045, 2.147±0.067 and 2.533±0.055 (t=22.831, 7.280 and 26.220, P<0.05); The protein expression of miR-627 mimic+IGF-Ⅰ group was 2.477±0.102, 1.760±0.046, 2.387±0.049, which was significantly higher than that of miR-627 mimic group (t=3.83, 8.286, 3.436, P<0.05). Conclusion: miR-627 can inhibit the proliferation of Fbs in hypertrophic scar by targeting IGF-Ⅰ.