基于TCGA数据库的食管鳞状细胞癌差异表达焦亡相关基因的筛选及GSDMC基因的验证  

Screening of differentially expressed pyroptosis-related genes in esophageal squamous cell carcinoma based on TCGA database and validation of GSDMC gene

作　　者：苗婷婷 胡浇浇 郑士耀 孙畅 杨铭 曹玉文 王良海[1,2] 胡建明 沈西华[2] 杨兰 MIAO Ting-ting;HU Jiao-jiao;ZHENG Shi-yao;SUN Chang;YANG Ming;CAO Yu-wen;WANG Liang-hai;HU Jian-ming;SHEN Xi-hua;YANG Lan(Department of Pathology,School of Medicine,Shihezi University,Shihezi 832002,China;Department of Pathology,the First Affiliated Hospital,Shihezi University,Shihezi 832002,China)

机构地区：[1]石河子大学医学院病理学系,石河子832002 [2]石河子大学医学院第一附属医院病理科,石河子832002

出　　处：《临床与实验病理学杂志》2023年第2期132-138,共7页Chinese Journal of Clinical and Experimental Pathology

基　　金：国家自然科学基金(81860518);石河子大学国际科技合作推进计划(GJHZ202105)。

摘　　要：目的 通过对TCGA数据库中食管鳞状细胞癌(esophageal squamous cell carcinoma, ESCC)组织基因表达谱RNA测序数据进行生物信息学分析,探讨与ESCC预后有关的焦亡基因,并通过体外实验对筛选出的基因进行验证和功能研究。方法 下载TCGA和GTEx数据库中ESCC和正常食管样本中mRNA数据及临床数据。从文献中获得细胞焦亡相关基因(pyroptosis-related genes, PRG),使用R语言及DESeq2软件获取差异表达的PRG。采用Cox单因素、多因素回归分析,筛选出与预后相关的基因。应用qRT-PCR验证差异表达的PRG在ESCC和正常食管上皮细胞的表达水平;运用siRNA干扰ESCC细胞中已筛基因的表达,qRT-PCR检测siRNA的干扰效率。干扰成功后分别采用CCK-8、平板克隆和qRT-PCR实验,检测干扰已筛基因后ESCC细胞的活力、克隆形成能力和凋亡。运用肿瘤免疫评估资源(TIMER)数据库,分析gasdermin家族蛋白C(GSMDC)的mRNA表达与食管癌免疫细胞浸润水平的相关性。结果 33个PRG中与ESCC相关的差异表达基因有23个,其中13个基因表达上调,10个基因表达下调(P<0.001)。Cox单因素回归分析显示:CASP5和GSDMC基因与ESCC患者预后相关(P<0.01)。Cox多因素回归分析显示:GSDMC基因是ESCC患者预后相关的独立危险因素(P<0.01)。qRT-PCR结果显示:GSDMC基因的mRNA水平在ESCC细胞中上调(P<0.05)。敲低GSDMC基因可抑制ESCC细胞增殖[第4天OD值:(1.73±0.07)vs(1.10±0.09),t=13.55,P<0.001]和克隆形成能力[克隆形成数目:(686±94.30)vs(377±77.31),t=4.389,P<0.05],促进ESCC细胞凋亡(P<0.001)。TIMER数据库分析:GSDMC基因的表达水平与B细胞(r=-0.317)、CD8+T细胞(r=-0.15)、巨噬细胞(r=-0.252)浸润程度呈负相关(P均<0.001),与树突状细胞(r=0.353)的浸润水平呈正相关(P<0.001)。结论 GSDMC在ESCC组织中表达上调,可作为评估ESCC患者预后不良的生物学指标。Purpose Through bioinformatics analysis of gene expression profiling RNA sequencing data of esophageal squamous cell carcinoma(ESCC) from TCGA database, they explored the pyroptosis-related genes related to the prognosis of ESCC, and the screened genes were further verified and functional studied through in vitro experiments. Methods The mRNA data of ESCC, normal esophageal samples, and clinical data of esophageal cancer patients were downloaded from TCGA and GTEx databases. Pyroptosis-related genes(PRG) were obtained from existing literatures. Differentially expressed PRG were obtained by R language and DESeq2 software. Univariate and multivariate Cox regression analyses were performed to screen the genes associated with prognosis. qRT-PCR was used to detect the expression levels of differentially expressed PRG gene related to ESCC prognosis in human ESCC cells and human normal esophageal epithelial cells. Next, siRNA was used to interfere with the expression of the screened gene in ESCC cells, and qRT-PCR was used to detect the interference efficiency of siRNA. After yielding the above interference successfully, CCK-8, plate cloning and qRT-PCR were used to investigate the viability, clonogenesis and apoptosis of ESCC cells on the screened gene with succeeded interference, respectively. The correlation between the mRNA expression of gasdermin family proteins C(GSMDC) and the level of immune cell infiltration in esophageal cancer was analyzed using tumor immune evaluation resource(TIMER) database. Results 23 differentially expressed genes related to ESCC were found among 33 pyroptosis related genes, among which 13 genes were up-regulated and 10 genes were down-regulated(P<0.001). Univariate cox regression analysis showed that CASP5 and GSDMC genes were associated with the prognosis of ESCC patients(P<0.01). Multivariate cox regression analysis showed that GSDMC gene was an independent prognostic factor in ESCC patients(P<0.01). qRT-PCR showed that the mRNA level of GSDMC gene was up-regulated in ESCC cells(P<0.0

关 键 词：食管肿瘤 鳞状细胞癌 细胞焦亡 生物信息学 预后 免疫细胞 

分 类 号：R735.1[医药卫生—肿瘤]