miR-16-5p通过靶向SPARC促进人瘢痕疙瘩成纤维细胞凋亡的研究  

MiR-16-5p promotes the apoptosis of human keloid fibroblasts by targeting SPARC

作　　者：闫柄文 张仔昂 薛萍 YAN Bingwen;ZHANG Ziang;XUE Ping(Department of Plastic Surgery,Xijing Hospital,Air Force Medical University,Xi'an 710032,China)

机构地区：[1]空军军医大学西京医院整形外科,陕西西安710032

出　　处：《空军军医大学学报》2023年第7期655-660,共6页Journal of Air Force Medical University

基　　金：国家自然科学基金(82102355)。

摘　　要：目的 探究miR-16-5p对人瘢痕疙瘩成纤维细胞(KFB)凋亡的作用及机制。方法 利用实时PCR技术检测人正常皮肤成纤维细胞(NFB)和KFB中miR-16-5p水平;利用细胞转染分别将miR-16-5p的对照序列(对照组)、miR-16-5p的模拟物序列(模拟物组)、miR-16-5p的互补序列(抑制物组)转染入KFB后,利用荧光显像技术和实时PCR技术检测转染效果;利用流式细胞仪和形态学手段评估各组KFB凋亡情况;利用Western blotting技术检测各组KFB凋亡相关蛋白含量;利用荧光素酶报告基因技术和Western blotting技术验证miR-16-5p的下游靶基因。结果 与NFB相比,KFB中miR-16-5p低表达;与对照组相比,模拟物组KFB内miR-16-5p含量增加(P<0.05),抑制物组KFB内miR-16-5p含量降低(P<0.01);与对照组相比,模拟物组KFB凋亡增加,抑制物组KFB凋亡减少(均P<0.05);与对照组相比,模拟物组KFB中Caspase-3和Bax表达增加,而Bcl-2表达减少;抑制物组KFB中Caspase-3和Bax表达减少,而Bcl-2表达增加(均P<0.05);与对照组相比,模拟物组抑制富含半胱氨酸的酸性分泌蛋白基因(SPARC)3′UTR质粒的荧光素酶活性(P<0.05);与对照组相比,模拟物组KFB中SPARC蛋白低表达,而抑制物组KFB中SPARC蛋白高表达(P<0.05)。结论 miR-16-5p在KFB中低表达,miR-16-5p可通过靶向SPARC促进KFB的凋亡。Objective To investigate the role and mechanism of miR-16-5p on the apoptosis of human keloid fibroblasts(KFB).Methods The levels of miR-16-5p in human normal skin fibroblasts(NFB)and KFB were detected by real-time PCR.MiR-16-5p negative control sequence(control group),miR-16-5p mimic sequence(mimic group)and miR-16-5p complementary sequence(inhibitor group)were separately transfected into KFB by cell transfection.The transfection effect was detected by fluorescence imaging and real-time PCR.The apoptosis of KFB in each group was evaluated by flow cytometry and morphological method.The contents of KFB apoptosis-related proteins in each group were detected by Western blotting.Luciferase reporter gene assay and Western blotting were used to verify the downstream target genes of miR-16-5p.Results Compared with NFB,the expression of miR-16-5p was lower in KFB.Compared with the control group,the content of miR-16-5p in KFB of the mimic group increased(P<0.05),while that of the inhibitor group decreased(P<0.01).Compared with the control group,the apoptosis of KFB in the mimic group increased,while that in the inhibitor group decreased(all P<0.05).Compared with the control group,the expression of Caspase-3 and Bax in KFB increased,while the expression of Bcl-2 decreased in the mimic group.In the inhibitor group,the expressions of Caspase-3 and Bax decreased,while the expression of Bcl-2 increased(all P<0.05).Compared with the control group,the mimic group inhibited the luciferase activity of secreted protein acidic and rich in cysteine(SPARC)3′UTR plasmid(P<0.05).Compared with the control group,the expression of SPARC protein in KFB was lower in the mimic group,but higher in the inhibitor group(P<0.05).Conclusion MiR-16-5p is underexpressed in KFB.MiR-16-5p can promote the apoptosis of KFB by targeting SPARC.

关 键 词：人瘢痕疙瘩成纤维细胞 凋亡 微小RNA 富含半胱氨酸的酸性分泌蛋白 

分 类 号：R622[医药卫生—整形外科]