中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (12): 2492-2499.doi: 10.12307/2025.382

• 皮肤粘膜组织构建 skin and mucosal tissue construction • 上一篇    下一篇

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

夏童童1，马  芳1，2，孙浩原3，刘虹麟1，4，张正皓1，2，杨佳琪1，张慧萍5，吴  凯2，沈江涌6，姜怡邓1，李桂忠1，2   

  1. 1国家卫生健康委员会代谢性心血管疾病研究重点实验室，宁夏回族自治区银川市  750004；宁夏医科大学，2基础医学院，3临床医学院，4检验学院，宁夏回族自治区银川市  750004；5湖南省妇幼保健院医学遗传科，湖南省长沙市  410008；6宁夏医科大学总医院烧伤科，宁夏回族自治区银川市  750004

  • 收稿日期:2024-04-07 接受日期:2024-06-15 出版日期:2025-04-28 发布日期:2024-09-10
  • 通讯作者: 李桂忠，硕士，教授，国家卫生健康委员会代谢性心血管疾病研究重点实验室，宁夏回族自治区银川市 750004；宁夏医科大学基础医学院，宁夏回族自治区银川市 750004
  • 作者简介:夏童童，女，1997年生，河南省新乡市人，汉族，主要从事代谢性相关疾病分子机制研究。
  • 基金资助:
    国家自然科学基金项目(82370293，U21A20343)，项目负责人：姜怡邓；国家自然科学基金项目(82360628)，项目负责人：沈江涌；宁夏回族自治区重点研发计划重点项目(2023BEG02074)，项目负责人：姜怡邓；宁夏回族自治区重点研发计划重点项目(2021BEG02028)，项目负责人：吴凯；宁夏医科大学校级科研项目重点项目(XZ2022005)，项目负责人：姜怡邓

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

Xia Tongtong1, Ma Fang1, 2, Sun Haoyuan3, Liu Honglin1, 4, Zhang Zhenghao1, 2, Yang Jiaqi1, Zhang Huiping5, Wu Kai2, Shen Jiangyong6, #br# Jiang Yideng1, Li Guizhong1, 2#br#   

  1. 1NHC Key Laboratory of Metabolic Cardiovascular Diseases Research, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 2School of Basic Medical Sciences, 3School of Clinical Medicine, 4School of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 5Department of Medical Genetics, Hunan Maternal and Child Health Hospital, Changsha 410008, Hunan Province, China; 6Department of Burn, General Hospital of Ningxia Medical University, Ningxia Hui Autonomous Region, China
  • Received:2024-04-07 Accepted:2024-06-15 Online:2025-04-28 Published:2024-09-10
  • Contact: Li Guizhong, Master, Professor, NHC Key Laboratory of Metabolic Cardiovascular Diseases Research, Yinchuan 750004, Ningxia Hui Autonomous Region, China; School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • About author:Xia Tongtong, NHC Key Laboratory of Metabolic Cardiovascular Diseases Research, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Supported by:
    the National Natural Science Foundation of China, Nos. 82370293 and U21A20343 (to JYD) and 82360628 (to SJY); Key Project of Ningxia Hui Autonomous Region Key Research and Development Program, Nos. 2023BEG02074 (to JYD) and 2021BEG02028 (to WK); Key Project of Ningxia Medical University, No. XZ2022005 (to JYD)

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摘要：




文题释义：
RNA pull-down：通过体外转录法标记生物素RNA探针，然后与胞浆蛋白提取液孵育，形成RNA-蛋白质复合物，该复合物可与链霉亲和素标记的磁珠结合，从而与孵育液中的其他成分分离。复合物洗脱后，通过Western Blot实验检测特定的RNA结合蛋白是否与RNA相互作用，或者结合质谱筛选与RNA结合的未知蛋白。RNA pull-down技术作为研究RNA-蛋白互相作用的核心技术，已逐渐成为研究热点。 
增生性瘢痕：瘢痕是创伤愈合留下的痕迹，也是组织修复愈合的最终结果。在某些个体，修复过程发生异常可以导致组织过度增生而形成增生性瘢痕。目前增生性瘢痕的治疗方式多样，但最有效的治疗方式仍为手术，因此寻找新的治疗靶点具有重要临床意义。

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。
https://orcid.org/0009-0004-5859-569X(夏童童)

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

关键词: 增生性瘢痕, lncRNA HSFAS, 成纤维细胞, RNA pull-down, 质谱分析

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

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