微小RNA-205在人增生性瘢痕中的表达及作用

Expression and effect of microRNA-205 in human hypertrophic scar

目的探讨微小RNA-205(miR-205)在人增生性瘢痕中的表达及作用。方法采用实验研究方法。收集于2019年10月—2020年1月在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1例、女5例,年龄(36±7)岁]的增生性瘢痕组织及同时期于同单位就诊的6例符合入选标准的外伤患者[男2例、女4例,年龄(38±9)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-205和血小板反应蛋白1(TSP-1)的mRNA表达情况。取增生性瘢痕组织,培养第3~5代成纤维细胞(Fb),用于后续实验。取2批增生性瘢痕Fb,分成TSP-1+miR-205对照组、TSP-1+miR-205模拟物组和TSP-1突变+miR-205对照组、TSP-1突变+miR-205模拟物组,分别转染相应的序列。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肾荧光素酶的表达,并计算两者比值,以此表示TSP-1的活性。取2批增生性瘢痕Fb,分为miR-205对照组、miR-205模拟物组、miR-205抑制物组及miR-205对照组、miR-205模拟物组、miR-205模拟物+TSP-1组,分别转染相应的序列,转染后0(即刻)、12、24、36、48 h,用酶标仪检测细胞活力。取2批增生性瘢痕Fb,同细胞活力检测实验进行分组及处理,转染后24 h,行Hoechst 33258染色,观察细胞核皱缩情况,以此反映细胞凋亡情况。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、t检验及χ2检验。结果增生性瘢痕组织中miR-205的mRNA表达量为0.54±0.05,明显低于正常皮肤组织中的1.26±0.07(t=8.213,P<0.01)。增生性瘢痕组织中TSP-1的mRNA表达量为1.46±0.07,明显高于正常皮肤组织中的0.68±0.11(t=6.031,P<0.01)。转染后48 h,TSP-1+miR-205模拟物组细胞表示TSP-1活性的荧光素酶/肾荧光素酶比值为0.532±0.028,明显低于TSP-1+miR-205对照组的0.998±0.012(t=26.500,P<0.01);TSP-1突变+miR-205模拟物组细胞荧光素酶/肾荧光素酶比值为0.963±0.012,与TSP-1突变+miR-205对照组的0.976±0.010相近(t=0.816,P>0.05)。转染后12、24、36、48 h,miR-205模拟物组细胞活力明显低于miR-205对照组(t=6.169、12.670、27.130、12.670,P<0.05或P<0.01);转染后0、12、24、36、48 h,miR-205抑制物组细胞活力明显高于miR-205对照组(t=6.169、7.221、7.787、7.835、13.030,P<0.05或P<0.01)。转染后12、24、36、48 h,miR-205模拟物组细胞活力明显低于miR-205对照组和miR-205模拟物+TSP-1组(t=8.118、26.970、39.550、42.490,14.570、12.240、36.830、45.220,P<0.05或P<0.01)。转染后24 h,与miR-205对照组相比,miR-205模拟物组细胞凋亡增加,miR-205抑制物组细胞凋亡减少。转染后24 h,与miR-205模拟物组相比,miR-205对照组和miR-205模拟物+TSP-1组细胞凋亡减少。结论miR-205可以通过抑制TSP-1的表达,抑制人增生性瘢痕Fb的增殖,促进Fb的凋亡,可能成为增生性瘢痕潜在的治疗靶点。

Objective To investigate the expression and effect of microRNA-205(miR-205)in human hypertrophic scar.Methods The experimental research method was 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 reverse transcription 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.Two batches of hypertrophic scar Fbs were divided into TSP-1+miR-205 control group,TSP-1+miR-205 mimic group,and 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 grouped and treated as described in 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 three.Data were statistically analyzed with analysis of variance for factorial design,one-way analysis of variance,t test,and chi-square 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 and miR-205 mimic+TSP-1 group were decreased.Conclusions miR-205 can inhibit the proliferation and promote the apoptosis of Fbs in human hypertrophic scar by inhibiting the expression of TSP-1,which has the potential to be a therapeutic target for hypertrophic scar.