摘要
目的:探讨巨噬细胞帽状蛋白（CAPG）在食管鳞状细胞癌（ESCC）组织中的表达水平及其对ESCC细胞体外迁移、增殖、侵袭的影响以及相关机制的影响。方法:通过对来自基因表达数据库（GEO）的ESCC单细胞转录组数据进行综合分析，以揭示肿瘤间的异质性。整合和标准化GSE188900数据集的ESCC样本数据，采用主成分分析（PCA）对标准化数据进行降维和聚类，采用Single R算法注释细胞，使用inferCNV R包识别肿瘤细胞。最终确定食管上皮细胞为肿瘤细胞来源，并对高变基因进行基因集变异分析（GSVA）。随后，采用实时定量反转录聚合酶链反应（qRT-PCR）验证CAPG在ESCC组织样本和正常样本中的表达量。靶向CAPG的小片段干扰RNA（siRNA）转染人ESCC细胞EC1，以降低EC1细胞中CAPG的表达。通过体外功能实验验证CAPG对EC1细胞的增殖、迁移和侵袭等能力的影响。最后，通过qRT-PCR验证CAPG对ESCC细胞中上皮-间质转化（EMT）通路相关分子表达的影响。结果:通过对ESCC单细胞测序数据分析，发现食管上皮细胞为肿瘤细胞来源，而且EMT通路相关基因在食管上皮细胞中显著富集。将上皮细胞相关基因与EMT通路相关基因取交集得到核心基因：CAPG。与正常组织相比，ESCC组织中CAPG表达明显上调。其次，体外功能实验表明，CAPG敲低显著影响人ESCC细胞EC1的增殖、迁移和侵袭，而且CAPG敲除影响多个EMT相关分子的表达。最后，CAPG作为EMT通路相关基因的成员，促进ESCC中肿瘤细胞的克隆性增殖和转移。结论:CAPG在ESCC中高表达，具有促进ESCC细胞增殖、迁移和侵袭的作用；并且CAPG敲除影响多个EMT通路相关分子的表达。
Abstract
Objective:To investigate the expression level of macrophage-capping protein (CAPG) in esophageal squamous cell carcinoma (ESCC) and its effect on migration, proliferation and invasion of ESCC cells in vitro as well as the related mechanisms.Methods:Comprehensive analysis for single cell transcriptome data of ESCC from Gene Expression Omnibus (GEO) datasets was performed in order to reveal the tumor heterogeneity. After raw data from GSE188900 was integrated and standardized, Principal Component Analysis (PCA) was used for dimensionality reduction and clustering of the standardized data. Then, the Single R algorithm was used for cell annotation, and the inferCNV R package was used to identify tumor cells. Esophageal epithelial cells were finally identified as tumor cells and Gene Set Variation Analysis (GSVA) was performed on highly variable genes. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verified the expression level of hub gene in esophageal squamous cell carcinoma patient samples. Next, small interfering RNA (siRNA) targeting CAPG was transfected into human esophageal squamous cell carcinoma cell line EC1 in order to knock down the expression of CAPG in EC1 cells. In vitro functional assays was performed to verify the effect of CAPG on EC1 cells, including proliferation, migration, invasion and wound-healing assays. Finally, the effect of CAPG knockout on the expression of EMT pathway-related genes in esophageal squamous cell carcinoma cells was verified by qRT-PCR.Results:The single cell sequencing data of esophageal squamous cell carcinoma showed that esophageal epithelial cells were the source of tumor cells and EMT pathway-related genes were significantly enriched in esophageal epithelial cells. We intersected epithelial cells-related genes with EMT pathway-related genes to obtain the hub gene: CAPG. The expression of CAPG was significantly higher in esophageal squamous cell carcinoma tissues compared to normal tissues. In vitro functional experiments demonstrated that the knockout of CAPG markedly impeded the proliferation, migration and invasion abilities of esophageal squamous cell carcinoma cells. Furthermore, CAPG knockout influenced the expression of several genes associated with EMT pathway. Finally, CAPG acts as a member of EMT pathway-related genes and promotes clonal proliferation and metastasis of ESCC cells.Conclusions:In esophageal squamous cell carcinoma tissue, CAPG is significantly up-regulated and promotes the proliferation, migration, and invasion abilities of ESCC cells. Additionally, knockout of CAPG affects the expression of several genes related to EMT pathway.
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