长链非编码RNA SNHG1对增生性瘢痕成纤维细胞增殖的影响

Effect of long non-coding RNA-SNHG1 on the proliferation of hypertrophic scar fibroblasts

目的探讨长链非编码RNA核仁小RNA宿主基因1(lnc RNA SNHG1)对人增生性瘢痕成纤维细胞增殖的影响。方法纳入2019-2021年在宁夏医科大学总医院烧伤整形外科接受手术治疗的22例增生性瘢痕患者,收集其增生性瘢痕组织与瘢痕旁正常皮肤组织(瘢痕旁3 cm以内),从中分离培养成纤维细胞。采用q RT-PCR从组织和细胞水平检测SNHG1m RNA和mi R-382-3p相对表达水平。将增生性瘢痕成纤维细胞随机分为对照组、SNHG1阴性对照组(转染慢病毒空载体)、模拟物对照组(转染对照模拟物)、SNHG1阴性对照+模拟物对照组(转染慢病毒空载体和对照模拟物)、SNHG1过表达组(转染过表达慢病毒)、mi R-382-3p过表达组(转染mi R-382-3p模拟物)、SNHG1过表达+模拟物对照组(转染过表达慢病毒和对照模拟物)与SNHG1过表达+mi R-382-3p过表达组(转染过表达慢病毒和mi R-382-3p模拟物),采用q RT-PCR检测各组SNHG1、PCNA、p27 m RNA和mi R-382-3p的相对表达水平;CCK-8法检测各组细胞增殖能力;Ed U染色法检测各组细胞增殖水平;Western blotting检测各组p27和PCNA蛋白表达水平。结果与正常皮肤组织及其成纤维细胞相比,SNHG1 m RNA在增生性瘢痕组织(3.21±2.65 vs.1.14±0.61,P<0.001)及其成纤维细胞中呈高表达(0.91±0.08 vs.0.54±0.08,P<0.01),而mi R-382-3p表达下调(组织:0.53±0.34 vs.1.15±0.61,P<0.001;细胞:0.84±0.09 vs.1.01±0.004,P<0.05)。与SNHG1阴性对照组相比,SNHG1过表达组细胞增殖能力增强(0.23±0.03 vs.0.16±0.01,P<0.001),Ed U阳性细胞百分比明显增高(30.01%±5.70%vs.7.13%±4.40%,P<0.001),PCNA m RNA和蛋白相对表达水平增高(m RNA:2.97±0.33 vs.0.98±0.25,P<0.01;蛋白:2.20±0.09 vs.0.88±0.20,P<0.05),p27 m RNA和蛋白相对表达水平降低(m RNA:0.30±0.03 vs.1.42±0.15,P<0.001;蛋白:0.47±0.11 vs.1.13±0.19,P<0.05),mi R-382-3p相对表达水平降低(0.05±0.01vs.1.03±0.12,P<0.001);与SNHG1过表达+模拟物对照组相比,SNHG1过表达+mi R-382-3p过表达组细胞增殖能力减弱(0.15±0.02 vs.0.26±0.01,P<0.001),Ed U阳性细胞百分比下降(5.97%±0.33%vs.11.70%±0.87%,P<0.001),PCNA m RNA和蛋白相对表达水平降低(m RNA:0.64±0.09 vs.3.33±0.38,P<0.001;蛋白:1.70±0.36 vs.2.34±0.16,P<0.05),p27 m RNA和蛋白相对表达水平升高(m RNA:1.01±0.44 vs.0.09±0.04,P<0.05;蛋白:1.38±0.31 vs.0.50±0.09,P<0.05)。结论 SNHG1在增生性瘢痕中呈高表达,可负向调控mi R-382-3p的表达,进而促进增生性瘢痕成纤维细胞的增殖,有望成为治疗增生性瘢痕的新靶点。

Objective To investigate the effect of long non-coding small nucleolar RNA host gene 1 (lnc RNA-SNHG1) on the proliferation of human hypertrophic scar fibroblasts.Methods The hypertrophic scar tissue and normal skin tissue adjacent to the scar (within 3 cm near the scar) of 22 patients with hypertrophic scar treated and operated in the Department of Burn and Plastic Surger y of the General Hospital of Ning xia Medical University from 2019 to 2021 were collected,and human primar y fibroblasts were isolated and cultured from the hypertrophic scar and normal skin tissue adjacent to the scar,while the expression of SNHG1 and mi R-382-3p was detected at the tissue and primary cell levels by using q RT-PCR.Proliferative scar fibroblasts were randomly divided into control group,SNHG1 negative control group (adding empty lentivirus vectors),mimic control group (adding control mimic),SNHG1 negative control+mimic control group (adding empty lentivirus vectors and control mimic),SNHG1overexpression group (adding overexpression lentivirus),mi R-382-3p overexpression group (adding mi R-382-3p mimics),SNHG1overexpression+mimic control group (adding overexpression lentivirus and control mimics) and SNHG1 overexpression+mi R-382-3p overexpression group (adding overexpression lentivirus and mi R-382-3p mimics).q RT-PCR was used to detect the m RNA expression of SNHG1,PCNA,p27 and mi R-382-3p in each group;CCK-8 method to detect the proliferation viability of the cells in each group after transfection;Ed U staining method to detect the change of proliferation level of each group of cells after transfection;Western blotting to detect the expression levels of p27 and PCNA proteins in each group of cells after transfection.Results SNHG1 presented high expression in hypertrophic scar tissue (3.21±2.65 vs.1.14±0.61,P<0.001) and primary cells (0.91±0.08vs.0.54±0.08,P<0.01),whereas the expression of mi R-382-3p was down-regulated (0.53±0.34 vs.1.15±0.61,P<0.001;0.84±0.09vs.1.01±0.004,P<0.05).Compared with SNHG1 negative control group,the cell proliferation ability of SNHG1 overexpression group increased (0.23±0.03 vs.0.16±0.01,P<0.001),the percentage of Ed U positive cells significantly increased (30.01%±5.70%vs.7.13%±4.40%,P<0.001),the expression levels of PCNA m RNA and protein increased (m RNA:2.97±0.33 vs.0.98±0.25,P<0.01;protein:2.20±0.09 vs.0.88±0.20,P<0.05),the expression levels of p27 m RNA and protein decreased (m RNA:0.30±0.03vs.1.42±0.15,P<0.001;protein:0.47±0.11 vs.1.13±0.19,P<0.05),and the expression level of mi R-382-3p decreased significantly(0.05±0.01 vs.1.03±0.12,P<0.001).Compared with SNHG1 overexpression+mimic control group,the cell proliferation ability of SNHG1 overexpression+mi R-382-3p overexpression group decreased (0.15±0.02 vs.0.26±0.01,P<0.001),the percentage of Ed U positive cells decreased (5.97%±0.33%vs.11.70%±0.87%,P<0.001),the expression levels of PCNA m RNA and protein decreased significantly (m RNA:0.64±0.09 vs.3.33±0.38,P<0.001;protein:1.70±0.36 vs.2.34±0.16,P<0.05),the expression levels of p27 m RNA and protein increased (m RNA:1.01±0.44 vs.0.09±0.04,P<0.05;protein:1.38±0.31 vs.0.50±0.09,P<0.05).Conclusion SNHG1 presents high expression in hypertrophic scar and can negatively regulate mi R-382-3p expression to promote proliferation of primary hypertrophic scar fibroblasts,which may become a potential new target in the treatment of hypertrophic scar.