【研究目的】

颅面短小畸形（Craniofacial Microsomia, CFM）是最常见的先天性颅面畸形之一，大多为单侧发病，常见的临床表现包括下颌骨发育不全、小耳畸形、面神经麻痹和软组织缺陷等。该畸形累及部位众多，且表型复杂。目前研究表明母亲烟草暴露与CFM发病密切相关，但是具体致病机制尚不清楚。烟草烟雾是一种公认的致畸剂，研究表明它会增加胎儿CFM的患病风险。本文第一部分通过生物信息学筛选了CFM与吸烟和烟草烟雾污染（Tobacco Smoke Pollution，TSP）相关的潜在致病基因。本文第二部分应用转录组测序技术筛选烟草暴露的颅面短小畸形致病基因，并进一步通过生物信息学分析和细胞生物学特性实验，以验证筛选出的差异基因功能。在分子水平为CFM的发病机制提供新的证据。

【研究方法】

1. 截止2021年5月，分别以“hemifacial microsomia, oculo-auriculo-vertebral syndrome, goldenhar syndrome, gene, mutations”和“smoke，tobacco smoke pollution”为英文关键词，在PubMed数据库和毒理学基因组信息数据库(Comparative Toxicogenomics Database，CTD) 各自检索已知的CFM相关的致病基因和吸烟、TSP作用的靶点基因。将检索出的候选基因集采用致病基因分析、蛋白相互作用网络、构建、功能富集分析等多种生物信息学方法，筛选出吸烟及TSP与CFM发生的潜在致病基因。

2.纳入合并有烟草暴露风险因素的CFM患者12例及健康对照组4例，其中6例通过转录组测序技术筛选出合并母亲烟草暴露风险因素的CFM患者中特异性表达的基因，另外6例用于筛选出的关键分子进行实时荧光定量PCR（quantitative polymerase chain reaction, qPCR）检测进行测序结果验证。然后将敲减和过表达关键基因的质粒转染至脂肪间充质干细胞(Adipose-Derived Stem Cells, ADSC)进行细胞划痕愈伤、CCK8增殖、Tranwell迁移等测定细胞功能，验证差异表达基因对ADSC功能的影响。将转染后的ADSC进行成脂诱导，使用油红O染色和成脂指标CEBPα检测进一步评估差异表达基因对ADSC成脂能力的影响。

【研究结果】

1.基于公共数据库检索，在PubMed数据库中共筛选处CFM相关基因43个，在CTD数据库中筛选出13102个吸烟相关基因和12161个TSP相关靶点基因，根绝ToppGene筛选出各自排名前50位的基因。基因相关性和功能富集分析结果主要集中在组织形态发生和发育功能上。从蛋白互作网络中识别出2个聚类分数最高的模块，并筛选出10个节点基因：TP53、TRIM25、HNRNPL、HNRNPH1、APP、PLEKHA4、ESR2、RNF4、ESR1和ELAVL1。其中与吸烟和烟草烟雾诱导CFM 发生最相关的基因包括 TP53、ESR1、ESR2 和 HNRNPL。

2. 通过对母亲烟草暴露CFM患儿患侧面部与健康对照的脂肪组织进行RNA测序，共鉴定出333341个mRNA，其中1244个mRNA在两组间具有显著差异，其中上调基因710个，下调基因534个。其中，吸烟影响遗传标志物HNRNP所调控的成骨基因HOX，在患者样本中有差异性表达。基因富集分析表明，差异最显著的基因功能集中于颅面部的组织发育。根据功能富集分析和P值筛选出具有显著高表达的HOXB2、HAND2基因。使用qPCR验证结果提示CFM患者面部脂肪组织中HOXB2、HAND2表达明显增加，与RNA测序数据一致基因表达异常。

3. ADSC细胞功能实验显示过表达HAND2、HOXB2基因可显著减弱ADSC的迁移和增殖能力，而敲减两种基因可增强ADSC的细胞增殖和迁移。油红O染色验证显示HOXB2和HAND2基因过表达导致ADSC成脂能力减弱，基因敲减后细胞成脂能力增强。成脂指标CEBPα检测结果表明过表达上述基因后 CEBPα转录水平下降，基因敲减后CEBPα转录水平显著上升。

【结论】

1. 通过生物信息学分析筛选出了CFM与吸烟及TSP相关的重要节点基因、通路和模块。TP53、ESR1、ESR2 和 HNRNPL基因可能在吸烟及烟草烟雾污染诱导的CFM中发挥了重要作用。其中，HNRNPL基因亚家族参与RNA的多种生物学过程，从而影响RNA转录水平。且有文献表明HNRNP家族基因突变可通过刺激HOX基因表达影响颅面成骨及面部脂肪组织发育。基于既往研究，本研究假设“吸烟可能通过间接影响HOX基因表达导致CFM在内的颅面部畸形发生”。

2. 根据合并母亲烟草暴露风险因素的CFM患儿RNA测序结果分析，找到多种差异表达基因以及关键基因通路。结合基因通路富集分析和qPCR确定显著差异表达基因HOXB2和HAND2。上述两种基因在胚胎颅面系统发育起到重要作用。在患儿母亲烟草暴露的风险因素下，两种基因的高表达可能导致颅面部发育不良引起CFM相关表型的出现。

3. 根据过表达和敲减上述基因细胞功能实验，发现HOXB2和HAND2基因高表达导致ADSC细胞增殖、迁移和成脂能力明显减弱。细胞实验表明，上述基因表达异常可能导致面部软组织发育异常，为解释CFM病理生理过程提供新的思路。本研究可以为进一步探究吸烟所引起的CFM畸形分子机制提供了重要依据。后期实验将根据两种基因的调控网络和通路设计实验，进一步探索CFM骨骼、脂肪发育异常的发病机制。

【关键词】颅面短小畸形；颅颌面外科；风险因素；

【Research background】

Craniofacial Microsomia (CFM) is one of the most common congenital deformities affecting the craniofacial region, mostly occurring unilaterally and characterized by mandibular hypoplasia, ear malformations, facial nerve paralysis, and soft tissue deficiencies. The condition involves multiple anatomical sites and has a complex phenotype, but the specific pathogenic factors and genes involved in CFM are not yet fully understood. Tobacco smoke is a recognized teratogen that increases the risk of CFM in fetuses during pregnancy. The first part of this article analyzes potential pathogenic genes related to CFM and tobacco smoke pollution (TSP) by using bioinformatics analysis to screen for potential candidate genes. The second part applies transcriptome sequencing technology to explore the pathogenic genes of CFM, identifies differentially expressed genes highly associated with the CFM phenotype through bioinformatics analysis and cellular experiments to verify gene function, providing new evidence for the molecular mechanism of CFM at the molecular level.

【Materials and Methods】

1. Known CFM-related pathogenic genes and target genes of smoking and tobacco smoke pollution (TSP) were searched using the English keywords "hemifacial microsomia, oculo-auriculo-vertebral syndrome, goldenhar syndrome, gene, mutations" and "smoke, tobacco smoke pollution" in the PubMed database and the Comparative Toxicogenomics Database (CTD), respectively. The candidate gene set was then screened using various bioinformatics methods, such as pathogenic gene analysis, protein-protein interaction network construction, and functional enrichment, to identify potential pathogenic genes induced by smoking and TSP in CFM.

2. Six CFM patients with a history of maternal tobacco smoke exposure were included. Total RNA was extracted from facial adipose tissue of CFM patients with smoke exposure, and genes specifically expressed in smoke-exposed CFM patients were identified by transcriptome sequencing. The expression levels of key molecules were confirmed by quantitative polymerase chain reaction (qPCR). Knockdown and overexpression of key genes were performed by transfecting plasmids into adipose-derived stem cells (ADSC), and cell functions such as scratch wound healing, CCK-8 proliferation, and Tranwell migration were evaluated to validate the role of differentially expressed genes in ADSC function. The transfected ADSCs were then induced to differentiate into adipocytes. Oil Red O staining and CEBPα were used to evaluate the effects of differentially expressed genes on ADSC adipogenic capacity.

【Results】

1. Based on public databases, 43 CFM-related genes were screened in the PubMed database, 50 smoke and 50 TSP-related target genes were screened in the CTD database. The three known gene sets were subjected to correlation and functional enrichment analysis, which mainly focused on tissue morphogenesis and developmental functions. Two modules were identified from the protein interaction network, and 10 node genes were selected. The most relevant genes to the pathogenesis of CFM induced by smoking and tobacco smoke included TP53, ESR1, ESR2, and HNRNPL.

2. Through RNA sequencing of CFM-affected facial adipose tissue and healthy controls, a total of 333,341 mRNA transcripts were identified, of which 1,244 showed significant differences between the two groups, with 710 upregulated genes and 534 downregulated genes. The most significantly differentially expressed genes were enriched in the pathways related to craniofacial tissue development. HOXB2 and HAND2 genes were found to be significantly upregulated based on functional enrichment analysis and P value screening. qPCR validation confirmed the abnormal expression of these genes in CFM patient adipose tissue.

3. Functional experiments on ADSC after plasmid transfection showed that overexpression of HAND2 and HOXB2 genes significantly reduced ADSC migration and inhibited their proliferation. Conversely, knockdown of these genes enhanced ADSC proliferation and migration.

4. Oil Red O staining showed that overexpression of HOXB2 and HAND2 genes weakened ADSC adipogenesis, while knockdown of these genes enhanced adipogenesis. The trend in CEBPα, an adipogenesis marker, was consistent with the adipogenesis results. Overexpression of these genes significantly decreased CEBPα transcription levels, while knockdown significantly increased CEBPα transcription levels.

【Conclusion】

1. Important hub genes, pathways, and modules associated with smoking and tobacco smoke pollution-induced CFM were identified through bioinformatics analysis. Results suggest that TP53, ESR1, ESR2, and HNRNPL genes may play a significant role in smoke and tobacco smoke pollution-induced CFM. The HNRNPL gene subfamily participates in various biological processes of RNA, thereby affecting RNA transcription levels. Mutations in the HNRNP gene family may cause craniofacial deformities, including CFM, by stimulating HOX gene expression.

2. Based on RNA sequencing results, various differentially expressed genes and key gene pathways were found. Combined with pathway enrichment analysis and qPCR, HAND2 and HOXB2 were identified as significantly differentially expressed genes. The two genes play an important role in embryonic craniofacial system development. High expression of these genes under the risk factor of tobacco smoke exposure during pregnancy may lead to craniofacial malformation and the appearance of CFM-related phenotypes.

3. Cell functional experiments of overexpression and knockdown of the above genes showed that high expression of HAND2 and HOXB2 genes significantly reduces the proliferation, migration, and adipogenic capacity of ADSC. Cell experiments suggest that abnormal expression of these genes may lead to facial soft tissue development abnormalities, providing new ideas for explaining the pathological and physiological processes of CFM. This study provides an important basis for further exploring the molecular mechanism of CFM deformity. Subsequent experiments will design experiments based on the regulatory network and pathways of the two genes to further explore the pathogenesis of CFM.

【Key words】 Craniofacial Microsomia; Craniomaxillofacial Surgery; Risk Factor；