功能未知的lncRNA LINC00173影响增生性瘢痕形成的作用与机制研究

A long non-coding RNA (lncRNA) with unknown function, named as LINC00173, which is identified by our lncRNA sequencing screening, is suggested to be associated with hypertrophic scar. Our previous work is described as follow: quantitative RT-PCR demonstrated that LINC00173 was significantly over-expressed in hypertrophic scar tissues compared with adjacent normal tissue. Overexpression of LINC00173 increased collagen production. Moreover, bioinformatic analysis and RIP-PCR experiment revealed that LINC00173 could bind with the transcriptional co-activating protein p300/CBP so as to gain an epigenetically regulating potential. Expression microarray and in vitro validation suggested that overexpression of LINC00173 significantly up-regulated the expression of a fibroproliferative gene of hypertrophic scar, Flightless I, which is also a target gene of p300/CBP. Based on above results, we therefore hypothesized that LINC00173 might increase collagen synthesis by recuiting p300/CBP and subsequently increasing the expression of Flightless I. In this proposal, firstly, we plan to clarify the functional role of LINC00173 in hypertrophic scar formation. To achieve this goal, we design to over-express and knock-down LINC00173 in vitro (fibroblast cell) and evaluate the impact of LINC00173 on the abilities of collagen synthesis. Secondly, we aim to explore how LINC00173 exerts its regulatory function. Highlighted by the relationship between Flightless I gene and LINC00173, we design to investigate the mutual interaction between Flightless I and LINC00173 via RIP, RNA pull-down, ChIP, ChIRP, deletion mapping, mutagenesis and rescue experiments. Finally, we design to evaluate the expression level of LINC00173, CBP and Flightless I in a high throughput human hypertrophic scar tissue microarray, as well as their correlation with clinical profiles. Our study is the original source of innovation and can provide new potential biomarkers for the prevention and treatment of hypertrophic scar.

LINC00173（简称LINC）是我们通过测序技术筛选获得、高表达于增生性瘢痕组织、物种中高度保守、功能未知的lncRNA。前期发现：LINC过表达促进胶原合成、参与瘢痕增生；软件分析与实验提示，LINC与转录辅激活物p300/CBP结合，而p300/CBP可结合促瘢痕增生分子FLII基因的启动子；表达谱芯片与实验证实LINC过表达致FLII表达显著上调，因此我们提出LINC招募p300/CBP表遗传调控FLII促进胶原合成、参与瘢痕增生的假说。研究拟采用过表达/沉默策略，论证LINC在胶原合成、瘢痕增生中的作用；应用RIP、RNA pull-down、ChIP、染色体构象捕获、ChIRP、缺失定位、突变等，论证LINC招募p300/CBP结合FLII启动子、促进转录致胶原合成、参与瘢痕增生的分子机制。LINC功能与机制未见报道，研究可能为增生性瘢痕的防治提供新靶标。

增生性瘢痕，是皮肤创面愈合过程中胶原局部过度沉积而形成的突出于皮肤表面的增殖性瘢痕组织，是烧伤、外伤和术后常见问题。目前，虽然有手术治疗、激素注射以及局部加压等方法治疗瘢痕，但均存在局限性或不良并发症，至今仍然缺乏有效的防治手段。近年来，lncRNA在瘢痕形成中的作用备受关注。LINC00173含有2个转录本，已被报道在多个疾病中发挥功能，尚未见在瘢痕中的研究报道。本研究首先通过qPCR检测LINC00173两个转录本在瘢痕真皮成纤维细胞中的表达情况，结果显示TSV1、TSV2在瘢痕真皮成纤维细胞中均表达上调。进一步通过在瘢痕真皮成纤维细胞中过表达LINC00173 TSV1、TSV2，发现过表达LINC00173 TSV1之后，细胞增殖无显著变化，胶原基因COL1A1、COL1A2、COL3A1以及α-SMA的表达均上调；而过表达LINC00173 TSV2之后，细胞增殖显著降低，胶原基因COL1A1、COL1A2以及α-SMA的表达均下调；而TSV1与TSV2均显著促进细胞凋亡。进一步运用RNA-seq技术筛选出差异表达的下游基因，KEGG pathway分析以及GSEA基因富集分析发现LINC00173过表达之后可能与MAPK、PI3K/Akt以及Wnt信号通路存在相关性；Western blot实验检测发现过表达LINC00173之后，β-catenin蛋白的表达显著升高；进一步徐运用siRNA干扰ctnnb1的表达即敲降β-catenin蛋白，可显著减弱LINC00173对瘢痕真皮成纤维细胞凋亡的促进作用。这些结果表明LINC00173的两个转录本TSV1与TSV2通过促进β-catenin蛋白的表达促进细胞凋亡来发挥功能，而TSV1与TSV2对细胞增殖、胶原及α-SMA的影响可能存在不同的调控机制，仍需进一步挖掘。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的研究线索。