circNBPF1通过吸附miR-31、miR-199a调控皮肤增生性瘢痕成纤维细胞增殖和胶原蛋白表达的机制研究

The increased proliferation of skin fibroblasts and collagen synthesis are the pathological basis of hypertrophic scar (HS). It is of great significance to explore the regulatory mechanism of HS. In our earlier studies, circRNAs has been reported to regulate the expression of type I collagen expression in skin fibroblasts by sponging miRNA. However, how circRNA affects the proliferation of fibroblasts and the expression of collagen in HS is not clear. Recently, we also found that circNBPF1, which is the most closely related to HS, harbours the binding sites of miR-31 and miR-199a, which could regulate cell proliferation and collagen expression. We speculate that the expression of circNBPF1 in HS is down regulated, which reduce the inhibitory effects of miR-31/miR-199a on FIH and CAV-1, and ultimately promotes the proliferation viability and the expression of type I / III collagen in fibroblasts of HS. In this study, RNAi and DLR were used to explore the effect and mechanism of circNBPF1 on the proliferation and type I / III collagen expression in HS fibroblasts. The results will not only reveal the mechanism of HS, but also will benefit to the treatment and prevention of HS and related skin diseases.

成纤维细胞增殖过快、胶原蛋白合成增多是增生性瘢痕（HS）的病理基础，研究其调控机制对HS的防治有重要意义。我们前期已证实环状RNA(circRNA)可通过吸附miRNA调控皮肤成纤维细胞I型胶原的表达。但在HS中，环状RNA如何影响成纤维细胞增殖及胶原蛋白表达，诱发HS尚不清楚。我们已筛选出与HS最相关的circNBPF1，其在HS中表达下调，且具有可调控细胞增殖和Ⅰ/Ⅲ型胶原蛋白表达的miR-31、miR-199a的结合位点。我们推测HS中circNBPF1表达下调，解除了对miR-31/miR-199a的抑制，进而引起靶基因FIH、CAV-1水平上调，最终促进HS成纤维细胞增殖和Ⅰ/Ⅲ型胶原蛋白表达。本研究拟采用RNAi、DLR等方法探讨circNBPF1对HS成纤维细胞增殖和Ⅰ/Ⅲ型胶原蛋白表达的调控作用及机制。本研究结果可进一步揭示HS发生的机制，为HS及相关皮肤病防治提供新思路。

瘢痕疙瘩是一种皮肤纤维化疾病，表现为成纤维细胞增殖过度以及I、III型胶原蛋白合成增多。瘢痕疙瘩不但影响外观，且可出现病变部位的瘙痒、溃疡，甚至恶变。虽然瘢痕疙瘩被归为良性皮肤疾病，但其常表现出与恶性肿瘤相似的生物学特征，但其机制未明。故研究瘢痕疙瘩的发病机制有着重要意义。. 本研究探讨了circRNAs人皮肤成纤维细胞和皮肤瘢痕成纤维细胞中的表达情况，发现了411种差异表达的circRNAs。GO和KEGG途径富集分析表明，差异表达的circRNAs主要参与细胞凋亡、局灶性粘附、PI3K-Akt和代谢途径，可能调控瘢痕的发生发展。用定量实时聚合酶链反应（qRT-PCR）和Sanger测序筛选出最可能调控瘢痕疙瘩成纤维细胞的circRNA—hsa-circRNA-0008259。接着，本研究采用慢病毒过表达、高通量测序、蛋白质组学、双荧光素酶报告基因检测系统、qRT-PCR等方法，探讨了hsa-circRNA-0008259调控瘢痕成纤维细胞Ⅰ、Ⅲ型胶原的表达的机制。结果发现hsa-circRNA-0008259可能通过抑制miR-423-5p，进而上调CTSD表达水平，最终抑制瘢痕疙瘩成纤维细胞中I型基因蛋白的表达。. 综上所述，我们的研究发现hsa-circRNA-0008259在瘢痕疙瘩成纤维细胞中表达水平下调，其可能通过hsa-circRNA-0008259/miR-423-5p/CTSD轴起调控I型基因蛋白表达的作用。本研究结果可为阐述瘢痕疙瘩的发病机制提供理论依据，为瘢痕疙瘩的治疗提供新的靶点。