miR-425-5p靶向TGF-β1/Smad2信号通路减少小鼠增生性瘢痕组织胶原纤维沉积

doi:10.16352/j.issn.1001-6325.2023.03.393

基础医学与临床 ›› 2023, Vol. 43 ›› Issue (3): 393-401.doi: 10.16352/j.issn.1001-6325.2023.03.393

miR-425-5p靶向TGF-β1/Smad2信号通路减少小鼠增生性瘢痕组织胶原纤维沉积

梁艳¹, 王丽娟¹, 吴惠林^2*

1.西安交通大学第一附属医院皮肤性病科,陕西西安 710061;
2.西安医学院第二附属医院皮肤科,陕西西安 710038

收稿日期:2022-02-23 修回日期:2022-07-25 出版日期:2023-03-05 发布日期:2023-02-27
通讯作者: * 790272640@qq.com
基金资助:
陕西省自然科学基础研究计划(2021JM-267)

miR-425-5p targets TGF-β1/Smad2 signaling pathway to attenuate collagen fiber deposition in hypertrophic scar tissue in mice

LIANG Yan¹, WANG Lijuan¹, WU Huilin^2*

1. Department of Dermatology and Venereology, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061;
2. Department of Dermatology,the Second Affiliated Hospital of Xi'an Medical University, Xi'an 710038, China

Received:2022-02-23 Revised:2022-07-25 Online:2023-03-05 Published:2023-02-27
Contact: * 790272640@qq.com

摘要/Abstract

摘要： 目的探究miR-425-5p在增生性瘢痕(HS)形成中的作用及机制。方法通过RT-qPCR和Western blot检测了16对HS组织和正常皮肤组织中miR-425-5p、转化生长因子-β1(TGF-β1)和SMAD家庭成员2(Smad2)的水平。将成纤维细胞分为9组:对照组(control组)、miR-425-5p-mimic组、NC-mimic组、miR-425-5p-inhibitor组、NC-inhibitor组、TGF-β1-pcDNA3.1组、NC-pcDNA3.1组、miR-425-5p-mimic+NC-pcDNA3.1组和miR-425-5p-mimic+TGF-β1-pcDNA3.1组,并采用Lipofectamine 2000对细胞进行转染处理。通过双荧光素酶报告基因实验验证miR-425-5p和TGF-β1的靶向关系。通过博莱霉素(BLM)诱导小鼠增生性瘢痕,然后将小鼠随机分为3组,对照组(NC-agomir)、模型组(NC-agomir+BLM)和转染miR-425-5p干预模型组(miR-425-5p-agomir+BLM),小鼠均治疗21 d。通过Masson三色染色检测HS组织的胶原纤维沉积。通过RT-qPCR和Western blot检测组织和细胞中miR-425-5p、Ⅰ 型胶原(Col-Ⅰ)、Ⅲ型胶原(Col-Ⅲ)、α-平滑肌肌动蛋白(α-SMA)、TGF-β1和Smad2的表达水平。结果与正常组相比,HS组的miR-425-5p表达水平降低(t=4.242,P＜0.001),而TGF-β1 mRNA和蛋白表达水平均升高(P＜0.001)。与对照组相比,miR-425-5p-mimic组的Col-Ⅰ、Col-Ⅲ、α-SMA、TGF-β1和Smad2的mRNA和蛋白表达水平均降低(P＜0.05)。与control组相比,miR-425-5p-inhibitor组的Col-Ⅰ、Col-Ⅲ、α-SMA、TGF-β1和Smad2的mRNA和蛋白表达水平均升高(P＜0.05)。与miR-425-5p-mimic共转染后,TGF-β1-3′-UTR-WT组的相对荧光素酶活性比TGF-β1-3′-UTR-MUT组降低(P＜0.05)。与miR-425-5p-mimic+NC-pcDNA3.1组相比,miR-425-5p-mimic+TGF-β1-pcDNA3.1组的Col-Ⅰ、Col-Ⅲ、α-SMA、TGF-β1和Smad2的mRNA和蛋白表达水平均升高(P＜0.05)。与NC-agomir+BLM组相比,miR-425-5p-agomir+BLM组的相对胶原蛋白含量降低(P＜0.05),miR-425-5p表达水平升高,而TGF-β1的mRNA和蛋白表达水平均降低(P＜0.05)。结论 miR-425-5p在HS组织中表达下调,上调miR-425-5p通过抑制TGF-β1/ Smad2信号通路抑制胶原沉积和HS形成。

关键词: 增生性瘢痕, miR-425-5p, 胶原沉积, 转化生长因子-β1, Smad2

Abstract: Objective To investigate the role and mechanism of miR-425-5p in the formation of hypertrophic scar(HS).Methods The levels of miR-425-5p, transforming growth factor-β1 (TGF-β1) and SMAD family member 2 (Smad2) in 16 pairs of HS tissue and normal skin tissue were detected by RT-qPCR and Western blot. The fibroblast cells were divided into nine groups: control group, miR-425-5p-mimic group, NC-mimic group, miR-425-5p-inhibitor group, NC-inhibitor group, TGF-β1-pcDNA3.1 group, NC-pcDNA3.1 group, miR-425-5p-mimic+NC-pcDNA3.1 group and miR-425-5p-mimic+TGF-β1-pcDNA3.1 group. The cells were transfected with Lipofectamine 2000. The targeting relationship between miR-425-5p and TGF-β1 was verified by dual-luciferase reporter gene assay. Hypertrophic scars in mice were induced by bleomycin(BLM), and then the mice were randomly divided into 3 groups, control group (NC-agomir), model group (NC-agomir+BLM) and intervention model group transfected with miR-425-5p (miR-425-5p-agomir+BLM), mice were treated for 21 days. Collagen fiber deposition in HS tissue was detected by Masson's trichrome staining. The expressions of miR-425-5p, type Ⅰ collagen (Col-Ⅰ), type Ⅲ collagen (Col-Ⅲ), α-smooth muscle actin (α-SMA), TGF-β1 and Smad2 in tissues and cells were detected by RT-qPCR and Western blot. Results Compared with normal group, the expression of miR-425-5p in HS group was decreased (t=4.242, P＜0.001), while the mRNA and protein expression of TGF-β1 was increased(P＜0.001). Compared with control group, the mRNA and protein expression of Col-Ⅰ, Col-Ⅲ, α-SMA, TGF-β1 and Smad2 in miR-425-5p-mimic group were all decreased (P＜0.05). Compared with control group, mRNA and protein expression of Col-Ⅰ, Col-Ⅲ, α-SMA, TGF-β1 and Smad2 in miR-425-5p-inhibitor group was all increased(P＜0.05). After co-transfection with miR-425-5p-mimic, the relative luciferase activity of TGF-β1-3′-UTR-WT group was reduced as compared with TGF-β1-3′-UTR-MUT group (P＜0.05). Compared with miR-425-5p-mimic+NC-pcDNA3.1 group, mRNA and protein expression of Col-Ⅰ, Col-Ⅲ, α-SMA, TGF-β1 and Smad2 in miR-425-5p-mimic+TGF-β1-pcDNA3.1 group were all increased (P＜0.05). Compared with NC-agomir+BLM group, the relative collagen content of miR-425-5p-agomir+BLM group was decreased (P＜0.05) and the expression of miR-425-5p was increased (P＜0.05). The TGF-β1 mRNA and protein were decreased (P＜0.05).Conclusions The miR-425-5p is down-regulated in HS tissue, and up-regulated miR-425-5p inhibits collagen deposition and HS formation by inhibiting TGF-β1/Smad2 signaling pathway.

Key words: hypertrophic scar, miR-425-5p, collagen deposition, transforming growth factor-β1, Smad2

中图分类号:

R619.6

梁艳, 王丽娟, 吴惠林. miR-425-5p靶向TGF-β1/Smad2信号通路减少小鼠增生性瘢痕组织胶原纤维沉积[J]. 基础医学与临床, 2023, 43(3): 393-401.

LIANG Yan, WANG Lijuan, WU Huilin. miR-425-5p targets TGF-β1/Smad2 signaling pathway to attenuate collagen fiber deposition in hypertrophic scar tissue in mice[J]. Basic & Clinical Medicine, 2023, 43(3): 393-401.

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miR-425-5p靶向TGF-β1/Smad2信号通路减少小鼠增生性瘢痕组织胶原纤维沉积

miR-425-5p targets TGF-β1/Smad2 signaling pathway to attenuate collagen fiber deposition in hypertrophic scar tissue in mice

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