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-βR13′-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。
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-βreceptor(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 typeⅠ&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 typeⅠ&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-βR13′-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 typeⅠ&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.
作者
王立铨
黄久佐
俞楠泽
马旭达
李天浩
龙笑
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)
出处
《基础医学与临床》
2022年第12期1841-1849,共9页
Basic and Clinical Medicine
基金
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)。
