circRNA_0008259通过抑制miR-423-5p调控瘢痕疙瘩成纤维细胞Ⅰ型胶原蛋白的表达

circRNA_0008259 Regulates the Expression of Type Ⅰ Collagen in Keloid Fibroblasts via Inhibiting miR-423-5p

目的探讨circRNA_0008259调控瘢痕疙瘩成纤维细胞(KFs)Ⅰ型胶原蛋白表达的机制。方法采用慢病毒过表达circRNA_0008259,分别应用高通量测序及蛋白质组学研究过表达circRNA_0008259后KFs中miRNAs和蛋白质的变化。通过生物信息学以及qRT-PCR分析验证circRNA_0008259下游的靶miRNA和蛋白。应用双荧光素酶报告基因检测法(DLR)验证circRNA_0008259与下游靶miRNA的结合。结果过表达circRNA_0008259后差异表达显著的miRNAs有48种(倍数≥2,P<0.05),差异表达的蛋白有40种(倍数≥1.3,P<0.05)。qRT-PCR证实miR-423-5p水平显著下调,DLR证实miR-423-5p可与circRNA_0008259及靶基因CTSD结合。结论circRNA_0008259可能通过抑制miR-423-5p,进而上调CTSD表达水平,最终抑制KFs中Ⅰ型胶原蛋白的表达。

Objective To explore the mechanism of the regulation role of circRNA_0008259 on the expression of type Ⅰ collagen in keloid fibroblasts(KFS).Methods Lentivirus was used to overexpress circRNA_0008259.High throughput sequencing and proteomics were used to investigate the miRNAs and proteins expression profile after overexpression of circRNA_0008259 in keloid fibroblasts(KFs).The downstream miRNAs and proteins of circRNA_0008259 were predicted and verified by bioinformatics and qRT-PCR.Double luciferase reporter gene assay(DLR) was used to verify the binding of cricRNA_0008259 and its downstream miRNA.Results 48 miRNAs(fold change≥2, P < 0.05) and 40 proteins were differentially expressed(fold change≥1.3, P < 0.05) in cricRNA_0008259 overexpressed KFs.qRT-PCR confirmed that the level of miR-423-5 p was significantly downregulated.DLR verified that miR-423-5 p could bind to circRNA_0008259 and the target gene CTSD(cathepsin D).Conclusion circRNA_0008259 may enhance the expression level of CTSD by inhibiting miR-423-5 p, and finally inhibit the expression of type Ⅰ collagen in KFs.