miR-2116-3p在人增生性瘢痕中的表达及作用  

Expression and effect of microRNA-2116-3p in human hypertrophic scar

作　　者：田文融 左俊 余扬[1] 齐郁松 艾江 卜盼盼 赵皎均 马志伟 李培培 马少林[1] Tian Wenrong;Zuo Jun;Yu Yang;Qi Yusong;Ai Jiang;Bu Panpan;Zhao Jiaojun;Ma Zhiwei;Li Peipei;Ma Shaolin(Department of Plastic Surgery,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,Xinjiang Uygur Autonomous Region,China)

机构地区：[1]新疆医科大学第一附属医院,新疆维吾尔自治区乌鲁木齐市830011

出　　处：《中国组织工程研究》2023年第28期4480-4486,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81760345):中医药提取物总黄酮抑制增生性瘢痕TGF-β1激活下游MAPK与Smad信号通路调控机制研究,项目负责人:马少林;新疆维吾尔自治区科研创新项目基金(XJ2919G202):肉苁蓉苯乙醇总苷对人增生性瘢痕成纤维细胞基于TGF-β/Smad信号通路的机制研究,项目负责人:艾江;新疆维吾尔自治区科研创新项目基金(XJ2022G174):miRNA在人增生性瘢痕中的表达及作用,项目负责人:田文融。

摘　　要：背景:目前多项研究证实了miRNA影响增生性瘢痕的发生发展,Ⅰ型胶原与增生性瘢痕成纤维细胞密切相关,猜测miR-2116-3p也有可能与增生性瘢痕的发生发展有关系。目的:探讨miR-2116-3p在人增生性瘢痕中的表达及作用。方法:收集新疆医科大学第一附属医院6例患者的增生性瘢痕组织与6例患者重睑术后正常皮肤组织,采用qRT-PCR法检测miR-2116-3p与Ⅰ型胶原的表达。取增生性瘢痕组织,原代培养第3-6代成纤维细胞,分为阴性对照组、miR-2116-3p模拟物组和miR-2116-3p抑制物组,分别转染对应的序列,用CCK-8法与EdU试剂盒检测细胞增殖活力,划痕实验检测细胞迁移能力,流式细胞术检测细胞凋亡,qRT-PCR法与Western blot法检测Ⅰ型胶原、Ⅲ型胶原和α平滑肌肌动蛋白的基因与蛋白表达,双荧光素酶实验验证其靶向结合。结果与结论:(1)增生性瘢痕组织中miR-2116-3p的mRNA表达量低于正常皮肤组织(P<0.01),Ⅰ型胶原的mRNA表达量低于正常皮肤组织(P<0.01);(2)转染后24,48,72 h,与阴性对照组比较,miR-2116-3p模拟物组细胞活力降低(P<0.05或P<0.01),miR-2116-3p抑制物组细胞活力升高(P<0.05或P<0.01);转染后48 h,与阴性对照组比较,miR-2116-3p模拟物组细胞EdU标记数减少(P<0.01),miR-2116-3p抑制物组EdU标记数增加(P<0.05);(3)转染后24 h,与阴性对照组比较,miR-2116-3p模拟物组细胞划痕面积、细胞凋亡率增加(P<0.01),miR-2116-3p抑制物组细胞划痕面积、细胞凋亡率减少(P<0.01或P<0.05);(4)转染后24h,与阴性对照组比较,miR-2116-3p模拟物组Ⅰ型胶原、Ⅲ型胶原和α-平滑肌肌动蛋白的基因与蛋白表达下降(P<0.05或P<0.01),miR-2116-3p抑制物组Ⅰ型胶原、Ⅲ型胶原和α-平滑肌肌动蛋白的蛋白表达升高(P<0.01);(5)双荧光素酶实验显示,miR-2116-3p能够与Ⅰ型胶原靶向结合;(6)结果表明,人增生性瘢痕中miR-2116-3p表达下调,miR-2116-3p可能通过靶向抑制Ⅰ型胶原的表达�BACKGROUND:A wide range of studies have demonstrated that microRNA(miR)plays a crucial role in hypertrophic scars development,and type Ⅰ collagen is closely related to hypertrophic scar fibroblasts.There might be a relationship between miR-2116-3p and occurrence and development of hypertrophic scars.OBJECTIVE:To investigate the expression and role of miR-2116-3p on human hypertrophic scar fibroblasts.METHODS:Hypertrophic scar tissues were collected from six patients from the First Affiliated Hospital of Xinjiang Medical University and normal skin tissues were collected from another six patients after blepharoplasty in the same unit at the same time.The expression of miR-2116-3p and type Ⅰ collagen was detected by qRT-PCR.Hypertrophic scar tissues were taken and primary cells were cultured to passages 3 to 6.Hypertrophic scar fibroblasts were divided into negative control group,miR-2116-3p mimic group,and miR-2116-3p inhibitor group,and transfected with corresponding sequences.Cell proliferation was detected by cell counting kit-8 and EdU kit.Cell migration was detected by cell scratch assay,and apoptosis was detected by flow cytometry.Expression of type Ⅰ collagen,type Ⅲ collagen andα-smooth muscle actin was detected by western blot and qRT-PCR at protein and mRNA levels,respectively.Dual luciferase reporter assay system was used to carry out targeting assays.RESULTS AND CONCLUSION:The mRNA expression of miR-2116-3p and type Ⅰ collagen in hypertrophic scar tissues was significantly lower than that in normal skin tissues(P<0.01).After 24,48,and 72 hours of transfection,the cell viability of the miR-2116-3p mimic group was significantly lower than that of the negative control group(P<0.05 or P<0.01),while the cell viability of miR-2116-3p inhibitor group was significantly higher than that of the negative control group(P<0.05 or P<0.01).At 48 hours after transfection,the number of EdU positive cells in the miR-2116-3p mimic group was significantly lower than that in the negative control group,while the nu

关 键 词：miR-2116-3p Ⅰ型胶原 增生性瘢痕 成纤维细胞 皮肤纤维化 

分 类 号：R446[医药卫生—诊断学] R318[医药卫生—临床医学] R62