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

doi:10.12307/2025.382

Abstract

Abstract: 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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: hypertrophic sca, lncRNA HSFAS, fibroblast, RNA pull-down, mass spectrometry

CLC Number:

Xia Tongtong, Ma Fang, Sun Haoyuan, Liu Honglin, Zhang Zhenghao, Yang Jiaqi, Zhang Huiping, Wu Kai, Shen Jiangyong, Jiang Yideng, Li Guizhong. Role and mechanism of long non-coding RNA HSFAS in hypertrophic scar analyzed using RNA pull-down combined mass spectrometry [J]. Chinese Journal of Tissue Engineering Research, 2025, 29(12): 2492-2499.

Figures/Tables 9

2.6 GO和KEGG富集分析与lncRNA HSFAS相互结合蛋白对相互结合的蛋白进行GO功能富集分析，GO分析主要包括生物过程、分子功能和细胞成分，结果显示：这些蛋白主要与RNA剪接和加工、染色体合成和分离、细胞周期等过程有关，选取前20个差异显著的通路绘制分类柱状图(图6A)。对相互结合的蛋白进行KEGG功能富集分析，发现这些相互结合的蛋白主要参与基因信息加工、细胞活动过程以及生化代谢途径，在基因信息加工分类中涉及RNA剪接、剪接体、RNA降解等；细胞活动涉及细胞衰老、细胞周期和坏死性凋亡；生化代谢途径主要涉及激素信号通路、胆固醇代谢和氨基酸合成等过程(图6B)。以上GO和KEGG富集分析结果表明，lncRNA HSFAS可能通过调控成纤维细胞中RNA的剪接和加工过程参与增生性瘢痕的发生。 2.7 支架附着因子B2和DICER1的质谱峰图通过对与lncRNA HSFAS相互结合的蛋白功能分析，选取了前10个结合蛋白中与RNA加工和剪接相关的蛋白支架附着因子B2和DICER1，发现了支架附着因子B2和DICER1与lncRNA HSFAS结合的特异性肽段(图7)。 2.8 生物信息学验证lncRNA HSFAS与支架附着因子B2和DICER1相互结合为进一步证实lncRNA HSFAS与支架附着因子B2和DICER1的相互结合，通过catRAPID(http://service.tartaglialab.com/page/catrapid_group)和RPISeq (http://pridb.gdcb.iastate.edu/RPISeq/)在线网站分析lncRNA HSFAS与支架附着因子B2和DICER1结合相对应的片段并预测结合概率，概率 > 0.50的预测被认为是有意义的。 catRAPID检测结果显示，支架附着因子B2和DICER1与lncRNA HSFAS存在多个相互结合位点(图8A)；RPISeq结果显示：lncRNA HSFAS与支架附着因子B2结合的随机森林和支持向量机分别为0.75和0.95；lncRNA HSFAS与DICER1结合的随机森林和支持向量机分别为0.7和0.99(图8B)。提示lncRNA HSFAS与支架附着因子B2和DICER1存在相互结合，进一步支持了质谱的检测结果。"

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