hsa-Let-7a-5p靶向结合TGF-βR1抑制局限性硬皮病成纤维细胞纤维化

doi:10.16352/j.issn.1001-6325.2022.12.1841

基础医学与临床 ›› 2022, Vol. 42 ›› Issue (12): 1841-1849.doi: 10.16352/j.issn.1001-6325.2022.12.1841

hsa-Let-7a-5p靶向结合TGF-βR1抑制局限性硬皮病成纤维细胞纤维化

王立铨, 黄久佐, 俞楠泽, 马旭达, 李天浩, 龙笑^*

中国医学科学院北京协和医学院北京协和医院整形外科,北京 100032

收稿日期:2022-09-26 修回日期:2022-10-21 出版日期:2022-12-05 发布日期:2022-11-23
通讯作者: ^* pumclongxiao@126.com
基金资助:
中国医学科学院医学与健康科技创新工程“十四五”项目中国罕见病的精准诊疗研究(2021-I2M-1-003);科技部战略性国际科技创新合作重点专项(2020YFE0201600);北京协和医院中央高水平医院临床科研专项青年培优计划(2022-PUMCH-A-210)

hsa-Let-7a-5p inhibits fibrosis of fibroblasts from localized scleroderma through targeted binding of TGF-βR1

WANG Li-quan, HUANG Jiu-zuo, YU Nan-ze, MA Xu-da, LI Tian-hao, LONG Xiao^*

Department of Plastic Surgery, Peking Union Medical College Hospital, CAMS & PUMC, Beijing 100032, China

Received:2022-09-26 Revised:2022-10-21 Online:2022-12-05 Published:2022-11-23
Contact: ^* pumclongxiao@126.com

摘要/Abstract

摘要： 目的探讨hsa-Let-7a-5p对局限性硬皮病成纤维细胞(LSFs)纤维化过程的影响及其与转化生长因子-β受体(TGF-βR)和TGF-β/Smad信号通路的关系。方法将携带hsa-Let-7a-5p和shTGF-βR1的慢病毒载体及空白慢病毒载体感染LSFs。CCK-8法和划痕实验检测各组LSFs的增殖和迁移情况。RT-qPCR、免疫荧光和Western blot检测转染的效率以及各组LSFs的Ⅰ、Ⅲ型胶原、α-SMA、TGF-βR1、TGF-β、p-Smad2/3的mRNA和蛋白表达水平。结果病毒感染LSFs后,shTGF-βR1组的TGF-βR1表达量明显低于对照组(P＜0.05);shTGF-βR1组的细胞增殖率、迁移能力均有所降低;shTGF-βR1组的Ⅰ、Ⅲ型胶原、α-SMA、TGF-β、p-Smad2/3的mRNA和蛋白表达水平均明显低于对照组(P＜0.05)。生物信息学分析表明hsa-Let-7a-5p可以与TGF-βR1 3′-UTR的靶区位置配对,hsa-Let-7a-5p慢病毒转染组的hsa-Let-7a-5p表达量明显高于对照组(P＜0.05),TGF-βR1表达量明显降低(P＜0.05)。hsa-Let-7a-5p组的细胞增殖率、迁移能力均有所降低;hsa-Let-7a-5p组的Ⅰ、Ⅲ型胶原、α-SMA、TGF-β、p-Smad2/3的mRNA和蛋白表达水平均明显低于对照组(P＜0.05)。结论 hsa-Let-7a-5p在体外通过靶向结合TGF-βR1调控TGF-β/Smad通路,抑制LSFs增殖与迁移,降低LSFs的纤维化标志物,从而抑制局限性硬皮病的纤维化。

关键词: hsa-Let-7a-5p, 局限性硬皮病, 纤维化

Abstract: Objective To investigate the effect of hsa-Let-7a-5p on the fibrosis process of localized scleroderma fibroblasts (LSFs) and its correlation with transforming growth factor-β receptor (TGF-βR) and TGF-β/Smad signaling pathway. Methods LSFs were infected with lentiviral vectors carrying hsa-Let-7a-5p and shTGF-βR1 and blank lentiviral vectors. The proliferation and migration of LSFs in each group were detected by CCK-8 method and scratch test. Real-time quantitative PCR (RT-qPCR), immunofluorescence and Western blot were used to detect the transfection efficiency, and the mRNA and protein levels of collagen type Ⅰ & Ⅲ, α-SMA, TGF-βR1, TGF-β, p-Smad2/3 in LSFs in each group. Results After virus infection of LSFs, the expression of TGF-βR1 in the shTGF-βR1 group was significantly lower than that in the control groups (P＜0.05); the cell proliferation rate and migration of shTGF-βR1 group were inhibited(P＜0.05); The mRNA and protein expression of collagen type Ⅰ & Ⅲ, α-SMA, TGF-β, p-Smad2/3 were significantly lower than those of the control groups(P＜0.05). Bioinformatics analysis showed that hsa-Let-7a-5p can be matched with the target region of TGF-βR1 3′-UTR, and the expression of hsa-Let-7a-5p in the lentivirus transfection group was significantly increased compared with the control groups(P＜0.05), the expression of TGF-βR1 was significantly decreased(P＜0.05). The cell proliferation rate and migration ability of the hsa-Let-7a-5p group were decreased; the mRNA and protein expression of collagen type Ⅰ & Ⅲ, α-SMA, TGF-β, p-Smad2/3 in the hsa-Let-7a-5p group were significantly lower than those of the control groups(P＜0.05). Conclusions hsa-Let-7a-5p regulates TGF-β/Smad pathway by targeting at TGF-βR1,inhibits proliferation and migration of LSFs, decreases fibrotic markers in LSFs thereby inhibiting fibrosis in localized scleroderma.

Key words: hsa-Let-7a-5p, localized scleroderma, fibrosis

中图分类号:

王立铨, 黄久佐, 俞楠泽, 马旭达, 李天浩, 龙笑. hsa-Let-7a-5p靶向结合TGF-βR1抑制局限性硬皮病成纤维细胞纤维化[J]. 基础医学与临床, 2022, 42(12): 1841-1849.

WANG Li-quan, HUANG Jiu-zuo, YU Nan-ze, MA Xu-da, LI Tian-hao, LONG Xiao. hsa-Let-7a-5p inhibits fibrosis of fibroblasts from localized scleroderma through targeted binding of TGF-βR1[J]. Basic & Clinical Medicine, 2022, 42(12): 1841-1849.

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hsa-Let-7a-5p靶向结合TGF-βR1抑制局限性硬皮病成纤维细胞纤维化

hsa-Let-7a-5p inhibits fibrosis of fibroblasts from localized scleroderma through targeted binding of TGF-βR1

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