Guo B Y, Lin F, Hui Q, Wang H Y
Department of Burns and Plastic Surgery, General Hospital of Northern Theater Command, Shenyang 110016, China.
Zhonghua Shao Shang Za Zhi. 2021 Apr 20;37(4):369-376. doi: 10.3760/cma.j.cn501120-20200225-00090.
To investigate the expression and effect of microRNA-627 (miR-627) in human hypertrophic scar. The experimental research method was used. From October 2019 to January 2020, hypertrophic scar tissue from 6 patients with hypertrophic scar (2 males and 4 females, aged (34±11) years) and the remaining normal skin tissue from 6 trauma patients (3 males and 3 females, aged (35±13) years) after flap transplantation were collected. The above-mentioned 12 patients were admitted to the General Hospital of Northern Theater Command and met the inclusion criteria. The mRNA expression of miR-627 was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The 3rd to 5th passages of fibroblasts (Fbs) were isolated from hypertrophic scar tissue and cultured for subsequent experiments after identification. Fbs from hypertrophic scar were divided into miR-627 negative control group, miR-627 mimic group, and miR-627 inhibitor group. The corresponding sequences were transfected respectively. At 0 (immediately), 12, 24, 36, and 48 h after transfection, the cell viability was detected by thiazolyl blue method; at 24 h after transfection, the apoptosis was detected by flow cytometry; at 24 h after transfection, the protein expression levels of insulin-like growth factor Ⅰ (IGF-Ⅰ), type Ⅰ collagen, and α smooth muscle actin (α-SMA) were detected by Western blotting. Two batches of Fbs from hypertrophic scar were used, one batch was divided into IGF-Ⅰ wild type+miR-627 negative control group and IGF-Ⅰ wild type+miR-627 mimic group, and the other batch was divided into IGF-Ⅰ mutant+miR-627 negative control group and IGF-Ⅰ mutant+miR-627 mimic group. The corresponding sequences were transfected respectively. At 48 h after transfection, the expressions 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 negative control group, miR-627 mimic alone group, and miR-627 mimic+IGF-Ⅰ group, and were transfected with the corresponding sequences respectively. At 24 h after transfection, the protein expression levels of IGF-Ⅰ, type Ⅰ collagen, and α-SMA were detected by Western blotting. 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, independent sample test, and chi-square test. The expression of miR-627 mRNA in hypertrophic scar tissue was 0.47±0.06, which was significantly lower than 1.12±0.23 in normal skin tissue (=15.090, <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 negative control group (=9.918, 34.370, 13.580, 61.550, <0.05 or <0.01); the cell viability of miR-627 inhibitor group was significantly higher than that of miR-627 negative control group (=4.722, 8.616, 13.330, 14.000, <0.05 or <0.01). At 24 h after transfection, compared with the apoptosis rate (8.42±0.47)% in miR-627 negative control group, (10.89±0.35)% in miR-627 mimic group was significantly higher (=7.301, <0.01), and (5.00±0.22)% in miR-627 inhibitor group was significantly lower (=11.510, <0.01). At 24 h after transfection, compared with the cell protein expressions of IGF-Ⅰ, type Ⅰ collagen, and α-SMA in miR-627 negative control group, those in miR-627 mimic group were significantly lower (=25.470, 5.282, 7.415, <0.01), and those in miR-627 inhibitor group were significantly higher (=15.930, 8.857, 9.763, <0.01). At 48 h after transfection, the luciferase/renal luciferase ratio of IGF-Ⅰ of cells in IGF-Ⅰ wild type+miR-627 mimic group was 0.463±0.061, which was significantly lower than 0.999±0.011 in IGF-Ⅰ wild type+miR-627 negative control group (=16.852, <0.01); the luciferase/renal luciferase ratio of IGF-Ⅰ of cells in IGF-Ⅰ mutant+miR-627 mimic group was 0.934±0.021, which was similar to 0.930±0.023 in IGF-Ⅰ mutant+miR-627 negative control group (=1.959, 0.05). At 24 h after transfection, the protein expressions of IGF-Ⅰ, type Ⅰ collagen, and α-SMA of cells in miR-627 mimic alone group were 1.623±0.070, 1.363±0.042, and 1.617±0.025, which were significantly lower than 2.723±0.045, 2.147±0.067, and 2.533±0.055 in miR-627 negative control group (=22.831, 7.280, 26.220, <0.01); the protein expressions of IGF-Ⅰ, type Ⅰ collagen, and α-SMA of cells in mimic+IGF-Ⅰ group were 2.477±0.102, 1.760±0.046, and 2.387±0.049, which were significantly higher than those of miR-627 mimic alone group (=3.830, 8.286, 3.436, <0.05 or <0.01). miR-627 expression in human hypertrophic scars is down-regulated; miR-627 can inhibit the proliferation and promote the apoptosis of Fbs in human hypertrophic scar by targeted inhibition of IGF-Ⅰ expression.
探讨微小RNA-627（miR-627）在人增生性瘢痕中的表达及作用。采用实验研究方法。收集2019年10月至2020年1月6例增生性瘢痕患者（男2例，女4例，年龄（34±11）岁）的增生性瘢痕组织以及6例皮瓣移植术后创伤患者（男3例，女3例，年龄（35±13）岁）剩余的正常皮肤组织。上述12例患者均入住北部战区总医院且符合纳入标准。采用实时荧光定量逆转录聚合酶链反应检测miR-627的mRNA表达。从增生性瘢痕组织中分离出第3至5代成纤维细胞（Fbs），鉴定后进行培养用于后续实验。将增生性瘢痕来源的Fbs分为miR-627阴性对照组、miR-627模拟物组和miR-627抑制剂组，分别转染相应序列。转染后0（即刻）、12、24、36和48 h，采用噻唑蓝法检测细胞活力；转染后24 h，采用流式细胞术检测细胞凋亡；转染后24 h，采用蛋白质印迹法检测胰岛素样生长因子Ⅰ（IGF-Ⅰ）、Ⅰ型胶原和α平滑肌肌动蛋白（α-SMA）的蛋白表达水平。采用两批增生性瘢痕来源的Fbs，一批分为IGF-Ⅰ野生型+miR-627阴性对照组和IGF-Ⅰ野生型+miR-627模拟物组，另一批分为IGF-Ⅰ突变型+miR-627阴性对照组和IGF-Ⅰ突变型+miR-627模拟物组，分别转染相应序列。转染后48 h，采用荧光素酶报告基因检测试剂盒检测荧光素酶和海肾荧光素酶的表达，并计算二者比值以反映IGF-Ⅰ的活性。将增生性瘢痕来源的Fbs分为miR-627阴性对照组、单独miR-627模拟物组和miR-627模拟物+IGF-Ⅰ组，分别转染相应序列。转染后24 h，采用蛋白质印迹法检测IGF-Ⅰ、Ⅰ型胶原和α-SMA的蛋白表达水平。细胞实验样本数为3。数据采用析因设计方差分析、单因素方差分析、独立样本t检验和卡方检验进行统计学分析。增生性瘢痕组织中miR-627 mRNA表达为0.47±0.06，显著低于正常皮肤组织中的1.12±0.23（F=15.090，P<0.01）。转染后12、24、36和48 h，miR-627模拟物组细胞活力显著低于miR-627阴性对照组（F=9.918、34.370、13.580、61.550，P<0.05或P<0.01）；miR-627抑制剂组细胞活力显著高于miR-627阴性对照组（F=4.
