摘要
目的研究长链非编码RNA(LncRNA)Ⅰ型胶原α2自然反义转录物1(COL1A2-AS1)对人瘢痕疙瘩成纤维细胞增殖、凋亡、迁移的影响。方法在COL1A2-AS1的功能研究中将人成纤维细胞分为3组:空白组、空载组、COL1A2-AS1-过表达组。qRT-PCR检测miR-181a-5p、TGF-β1、Smad7、Col-Ⅲ、COL1A2-AS1的表达。CCK-8法检测细胞增殖率,流式细胞术检测细胞凋亡,Transwell法检测细胞迁移,Western blot检测TGF-β1、Smad7、Col-Ⅲ蛋白的表达水平。双荧光素酶报告实验验证COL1A2-AS1与所预测的靶标miR-181a-5p的结合关系。结果与空载组比较,COL1A2-AS1-过表达组的人成纤维细胞增殖OD值、迁移细胞数以及Col-Ⅲ的表达都明显下调(P<0.05),而TGF-β1和Smad7的表达水平都明显升高(P<0.05)。与空载组比较,COL1A2-AS1-过表达组细胞的凋亡率增加(P<0.05)。另外,与空载组比较,COL1A2-AS1-过表达组明显抑制miR-181a-5p的表达(P<0.05),且经过双荧光素酶报告实验验证COL1A2-AS1和miR-181a-5p可直接结合。结论COL1A2-AS1直接靶向抑制miR-181a-5p的表达,并抑制人瘢痕疙瘩成纤维细胞的增殖和迁移,促进细胞的凋亡。
Objective To investigate the effects of long non-coding RNA(lncRNA)collagen type I alpha 2-antisense RNA 1(COL1a2-AS1)on the proliferation,apoptosis and migration of human keloid fibroblasts,and the underlying mechanism.Methods In the function study of COL1A2-AS1,human fibroblasts were treated with blank control,or transfected with negative control plasmid or COL1A2-AS1 overexpression plasmid.The expressions of microRNA-181a-5p(miR-181a-5p),transforming growth factor-β1(TGF-β1),small mothers against decapentaplegic 7(Smad7),typeⅢcollagen(ColⅢ)and COL1A2-AS1 were detected by Quantitative real-time polymerase chain reaction(qRT-PCR).Cell proliferation rate,apoptosis,and migration were detected by Cell Counting Kit-8(CCK-8)assay,flow cytometry and Transwell assay,respectively.Western blot was used to detect the expression levels of TGF-β1,Smad7,and ColⅢ.The binding relationship between COL1A2-AS1 and the predicted target gene miR-181a-5p was verified by dual-luciferase reporter assay.Results Compared with those transfected with negative control plasmid,human fibroblasts transfected with COL1A2-AS1 overexpression plasmid presented lower optical density(OD)value of proliferation rate,migratory cell number and expression level of ColⅢ(P<0.05),and higher expression levels of TGF-β1 and Smad7(P<0.05).In addition,overexpression of COL1A1-AS1 significantly increased the apoptotic rate(P<0.05)and downregulated miR-181a-5p(P<0.05).The direct binding relationship between COL1A2-AS1 and miR-181a-5p was verified by the dual-luciferase reporting assay.Conclusion COL1A2-AS1 directly targets the expression of miR-181a-5p,thus inhibiting the proliferation and migration of human keloid fibroblasts,and promoting cell apoptosis.
作者
詹明峰
张文娟
孙士芳
尚佩生
沈晓峰
ZHAN Mingfeng;ZHANG Wenjuan;SUN Shifang(Department of Dermatology,the Fifth Affiliated Hospital of Xinjiang Medical University,Xinjiang,Urumqi 830000,China)
出处
《河北医药》
CAS
2023年第17期2565-2570,共6页
Hebei Medical Journal
基金
新疆维吾尔自治区自然科学基金(编号:2020D01C226)。
