lncRNA8975-1：增生性瘢痕形成的新机制

Due to the limited and ineffective rescue tactics, hypertrophic scar becomes one difficult trouble in burn and plastic surgery. Thus to understand the mechanism of scar formation and search for new therapeutic targets have been hot spots in the field of wound healing. lncRNA8975-1 was specially over-expressed in hypertrophic scar, identified from differential lncRNA expression profile between hypertrophic scar and normal skin tissue. Previous study showed that overexpression of lncRNA8975-1 in normal fibroblast cells could increase collagen synthesis. On the other hand, bioinformatic analysis and experiment data indicated that lncRNA8975-1 could bind Smad3/CBP complex in the COL1A2 promoter. This study will confirm the role of lncRNA8975-1 in hypertrophic scar. Furthermore, in vitro transcription activity assay, promoter mutation, chromatin conformation capture, RIP and RNA pull down assay were used to demonstrate that lncRNA8975-1 recruits Smad3/CBP complex to the promoter of COL1A2, synthesizing more collagen, involving in hypertrophic scar formation. Up to now, there was no report about the function and mechanism of lncRNAs in regulating the hypertrophic scar. This study may offer a new theoretical basis to clarify hypertrophic scar pathology mechanism, and provide a new target for therapeutics of hypertrophic scar.

增生性瘢痕是烧伤、整形外科常见难题与研究热点。lncRNA8975-1是我们采用芯片技术筛选获得的、差异高表达于增生性瘢痕皮肤组织中的lncRNA。前期发现：lncRNA8975-1过表达可促进胶原合成；生物信息学与实验验证初步确定其通过招募Smad3/CBP复合物、特异性结合于COL1A2启动子而激活COL1A2表达，提示lncRNA8975-1可能通过靶向调节COL1A2的机制影响胶原合成和瘢痕增生。本研究拟：阐明lncRNA8975-1在增生性瘢痕形成中的生物学功能；以体外转录活性分析、启动子结合位点突变分析、染色体构象捕获分析、RIP、RNA pull down等技术，充分论证lncRNA8975-1招募Smad3/CBP复合物结合COL1A2启动子、促进转录致胶原合成、参与瘢痕增生形成的分子机制。研究有源头创新性，有助于阐明增生性瘢痕发生的机制，为其防治提供新线索和潜在干预靶标。

增生性瘢痕是烧伤外科和整形外科界面临的巨大挑战，皮肤损伤及外科手术后瘢痕的总体发病率为40％-70％，烧伤患者的发病率高达91％。其不仅影响美观，特殊部位的瘢痕还会影响患者的生活质量，给他们带来生理和心理的双重压力。目前，虽然有手术切除、压力治疗、局部皮质激素注射等方法治疗瘢痕，但是均存在局限性或不良并发症，无法从根本上阻止瘢痕产生。近年来，lncRNA不断被证实参与纤维化发生、细胞外基质降解等。我们采用lncRNA芯片技术筛选了人消退期瘢痕组织与成熟期瘢痕组织中差异lncRNA表达谱，其中一条功能未知的且差异高表达于消退期瘢痕组织的lncRNA8975-1，引起了我们的关注。我们通过qRT-PCR技术对15例增生性瘢痕患者的组织和原代分离的真皮成纤维细胞进行分析，结果显示与正常组相比瘢痕组织中lncRNA8975-1的表达水平均上调，与lncRNA芯片结果趋势一致。我们进一步在瘢痕真皮成纤维细胞中过表达lncRNA8975-1，qRT-PCR与Western blot结果显示胶原基因COL1A1、COL1A2、COL3A1以及α-SMA的表达均下调，CCK-8实验显示细胞增殖能力降低。而干扰lncRNA8975-1的表达之后，胶原基因COL1A1、COL1A2、COL3A1以及α-SMA的表达均上调，细胞增殖能力升高。lncRNA8975-1基因3’端RACE可以扩增出产物，长度约为653bp。核质分离与qRT-PCR实验显示lncRNA8975-1在细胞核与细胞质均有表达，且细胞核中表达较高。运用lncRNA8975-1 RNA pull down实验找出与lncRNA8975-1结合的蛋白，共鉴定到14个蛋白，过滤掉角蛋白等杂蛋白后，有9个蛋白质可供下一步研究。进一步在正常皮肤真皮成纤维细胞中过表达lncRNA8975-1，之后运用RNA-seq技术筛选出141个差异表达的下游基因，GO和KEGG pathway分析发现lncRNA8975-1过表达之后差异表达的基因主要与细胞生物合成过程、核酸结合、结合以及转录调节、代谢信号途径、MAPK信号途径等相关联。提示lncRNA8975-1可能通过影响MAPK信号通路等来发挥功能。这些结果有助于了解lncRNA作用于瘢痕增生的新机制，可能为解决瘢痕增生这一技术难题提供新靶标、新思路。