Expression and effect of microRNA-627 in human hypertrophic scar
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摘要:
目的 探讨微小RNA-627(miR-627)在人增生性瘢痕中的表达及作用。 方法 采用实验研究方法。收集2019年10月—2020年1月于北部战区总医院就诊的符合入选标准的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、同期于同单位就诊的6例符合入选标准的外伤患者[男3例、女3例,年龄(35±13)岁]行皮瓣移植手术后剩余的正常皮肤组织,采用实时荧光定量反转录PCR法检测miR-627的mRNA表达。取增生性瘢痕组织,培养第3~5代成纤维细胞(Fb),经鉴定后用于后续实验。取增生性瘢痕Fb,分为miR-627阴性对照组、miR-627模拟物组和miR-627抑制物组,分别转染对应的序列,转染后0(即刻)、12、24、36、48 h,用噻唑蓝法检测细胞活力;转染后24 h,用流式细胞术检测细胞凋亡情况;转染后24 h,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。取2批增生性瘢痕Fb,一批分为IGF-Ⅰ野生型+miR-627阴性对照组、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、IGF-Ⅰ突变型+miR-627模拟物组,分别转染对应的序列,转染后48 h,采用荧光素酶报告基因检测试剂盒分别检测荧光素酶和肾荧光素酶的表达,并计算两者比值,反映IGF-Ⅰ的活性。取增生性瘢痕Fb,分为miR-627阴性对照组、单纯miR-627模拟物组和miR-627模拟物+IGF-Ⅰ组,分别转染对应的序列,转染后24 h,采用蛋白质印迹法检测IGF-Ⅰ、Ⅰ型胶原和α-SMA蛋白表达水平。细胞实验中样本数均为3。对数据行析因设计方差分析、单因素方差分析、独立样本
t 检验及
χ 2检验。 结果 增生性瘢痕组织中miR-627的mRNA表达量为0.47±0.06,显著低于正常皮肤组织中的1.12±0.23(
t =15.090,
P <0.01)。转染后12、24、36、48 h,miR-627模拟物组细胞活力明显低于miR-627阴性对照组(
t =9.918、34.370、13.580、61.550,
P <0.05或
P <0.01),miR-627抑制物组细胞活力明显高于miR-627阴性对照组(
t =4.722、8.616、13.330、14.000,
P <0.05或
P <0.01)。转染后24 h,与miR-627阴性对照组的细胞凋亡率(8.42±0.47)%相比,miR-627模拟物组的(10.89±0.35)%显著升高(
t =7.301,
P <0.01),miR-627抑制物组的(5.00±0.22)%显著下降(
t =11.510,
P <0.01)。转染后24 h,与miR-627阴性对照组细胞IGF-Ⅰ、Ⅰ型胶原和α-SMA的蛋白表达相比,miR-627模拟物组明显下降(
t =25.470、5.282、7.415,
P <0.01),miR-627抑制物组明显升高(
t =15.930、8.857、9.763,
P <0.01)。转染后48 h,IGF-Ⅰ野生型+miR-627模拟物组细胞IGF-Ⅰ的荧光素酶/肾荧光素酶比值为0.463±0.061,明显低于IGF-Ⅰ野生型+miR-627阴性对照组的0.999±0.011(
t =16.852,
P <0.01);IGF-Ⅰ突变型+miR-627模拟物组细胞IGF-Ⅰ的荧光素酶/肾荧光素酶比值为0.934±0.021,与IGF-Ⅰ突变型+miR-627阴性对照组的0.930±0.023相近(
t =1.959,
P >0.05)。转染后24 h,单纯miR-627模拟物组细胞IGF-Ⅰ、Ⅰ型胶原和α-SMA的蛋白表达量1.623±0.070、1.363±0.042、1.617±0.025均较miR-627阴性对照组的2.723±0.045、2.147±0.067、2.533±0.055明显降低(
t =22.831、7.280、26.220,
P <0.01),miR-627模拟物+IGF-Ⅰ组细胞IGF-Ⅰ、Ⅰ型胶原、α-SMA的蛋白表达量2.477±0.102、1.760±0.046、2.387±0.049均较单纯miR-627模拟物组明显升高(
t =3.830、8.286、3.436,
P <0.05或
P <0.01)。 结论 人增生性瘢痕中miR-627表达下调;miR-627可通过靶向抑制IGF-Ⅰ的表达从而抑制人增生性瘢痕Fb的增殖,促进Fb的凋亡。
Abstract:Objective To investigate the expression and effect of microRNA-627 (miR-627) in human hypertrophic scar. Methods 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
t test, and chi-square test. Results 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 (
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 negative 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 negative control group (
t =4.722, 8.616, 13.330, 14.000,
P <0.05 or
P <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 (
t =7.301,
P <0.01), and (5.00±0.22)% in miR-627 inhibitor group was significantly lower (
t =11.510,
P <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 (
t =25.470, 5.282, 7.415,
P <0.01), and those in miR-627 inhibitor group were significantly higher (
t =15.930, 8.857, 9.763,
P <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 (
t =16.852,
P <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 (
t =1.959,
P> 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 (
t =22.831, 7.280, 26.220,
P <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 (
t =3.830, 8.286, 3.436,
P <0.05 or
P <0.01). Conclusions 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.
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Key words:
- Cicatrix /
- Fibroblasts /
- Insulin-like growth factor Ⅰ /
- MicroRNA-627
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