RNA pull-down联合质谱分析lncRNA HSFAS在增生性瘢痕中的作用及机制

Role and mechanism of long non-coding RNA HSFAS in hypertrophic scar analyzed using RNA pull-down combined mass spectrometry

背景:课题组前期研究发现,增生性瘢痕特异性长链非编码RNA HSFAS是一种可用于增生性瘢痕诊断的新型生物标志物,但其如何在增生性瘢痕中发挥作用尚不清楚。目的:探讨长链非编码RNA HSFAS在增生性瘢痕中的作用及机制。方法:临床收集3例行增生性瘢痕组织切除手术患者的新鲜增生性瘢痕皮肤组织和增生性瘢痕旁正常皮肤组织标本,应用免疫荧光技术检测两种皮肤组织冰冻切片中长链非编码RNA HSFAS的表达。应用酶消化法体外分离增生性瘢痕皮肤组织与正常皮肤组织原代成纤维细胞,采用qRT-PCR检测细胞中长链非编码RNA HSFAS mRNA表达,通过RNA pull-down联合质谱技术检测与长链非编码RNA HSFAS相互结合的蛋白,利用GO和KEGG分析长链非编码RNA HSFAS参与增生性瘢痕进展的主要功能和通路,通过catRAPID和RPISeq网站分析确定长链非编码RNA HSFAS与蛋白的靶向结合。结果与结论:①与正常皮肤组织相比,增生性瘢痕组织中的长链非编码RNA HSFAS表达升高(P<0.05);与正常皮肤组织来源成纤维细胞相比,增生性瘢痕组织来源成纤维细胞中长链非编码RNA HSFAS mRNA表达升高(P<0.05);②RNA pull-down联合质谱技术明确与长链非编码RNA HSFAS相互结合的蛋白有510个;GO和KEGG分析结果显示:这些蛋白主要涉及RNA剪接和加工、染色体合成和分离、细胞周期等过程,其中涉及RNA剪接和加工的蛋白有支架附着因子B2和DICER1,并且与长链非编码RNA HSFAS的结合分数较高;生物信息学技术分析验证结果显示,长链非编码RNA HSFAS与支架附着因子B2和DICER1蛋白存在相互结合;③结果显示,长链非编码RNA HSFAS可能通过与支架附着因子B2和DICER1蛋白相互结合调控RNA剪接和加工修饰影响基因表达,从而促进增生性瘢痕的发生发展。

BACKGROUND:Previous studies found that the proliferative scar-specific long non-coding RNA lncRNA HSFAS is a novel biomarker that can be used in the diagnosis of hypertrophic scar,but how it functions in hypertrophic scar is not clear.OBJECTIVE:To investigate the role and mechanism of lncRNA HSFAS in hypertrophic scar.METHODS:Fresh scar tissue and surrounding normal skin tissue samples from three patients with hypertrophic scar were collected,and tissue immunofluorescence was used to detect the expression of lncRNA HSFAS in frozen sections of two skin tissues.Primary fibroblasts were isolated from proliferative scarred skin tissue and normal skin tissue and cultured by enzyme digestion method.Quantitative real-time PCR was used to detect the mRNA expression of lncRNA HSFAS in cells.The proteins bound to lncRNA HSFAS were detected by RNA pull-down combined mass spectrometry.GO and KEGG were used to analyze the main functions and pathways of lncRNA HSFAS involved in hypertrophic scar progression.The targeted binding of lncRNA HSFAS to proteins was determined by catRAPID and RPISeq website analysis.RESULTS AND CONCLUSION:Compared with normal skin tissue and fibroblasts from normal skin tissue,the expression of lncRNA HSFAS in human hypertrophic scar tissue and primary fibroblasts from hypertrophic scar tissue was significantly increased(P<0.05).There were 510 proteins clearly bound to lncRNA HSFAS by RNA pull-down combined mass spectrometry.The results of GO and KEGG analyses showed that these proteins were mainly involved in RNA splicing and processing,chromosome synthesis and separation,and cell cycle.Among them,the proteins involved in RNA splicing and processing included scaffold attachment factor B2 and DICER1,and the binding fraction with lncRNA HSFAS was higher.The results of bioinformatics analysis showed that lncRNA HSFAS was bound to scaffold attachment factor B2 and DICER1 proteins.To conclude,lncRNA HSFAS may affect gene expression by interacting with scaffold attachment factor B2 and DICER1 proteins to regulate RNA splicing and processing modification,thus promoting the occurrence and development of hypertrophic scar.