基于高通量测序数据分析食管鳞状细胞癌circRNA的研究

Analysis of circRNA in esophageal squamous cell carcinoma based on high-throughput sequencing data

目的基于高通量测序数据分析食管鳞状细胞癌(ESCC)特异性差异表达的circRNA。方法选取空军军医大学唐都医院2018年3月至2019年3月病理确诊为ESCC的6例患者为研究对象,其中3例为Ⅰ期ESCC,3例为Ⅲ期ESCC。采用高通量测序技术对患者的癌组织和癌旁组织的circRNA表达情况进行差异性分析,对差异表达的基因进行GO富集分析、KEGG富集分析和维恩分析,利用Cytoscape软件构建circRNA-miRNA-mRNA网络。对癌组织中差异表达最显著的基因进行细胞学和组织学验证,并分析circRNA与患者临床病理特征的关系。结果在3例Ⅰ期ESCC患者的癌旁组织和癌组织中筛选出553个差异表达的circRNA,其中在癌组织中上调的有413个,下调的有140个;在3例Ⅲ期ESCC患者的癌旁组织和癌组织中筛选出425个差异表达的circRNA,其中在癌组织中上调的有276个,下调的有149个。GO富集分析显示,Ⅰ期ESCC患者差异表达circRNA的宿主基因主要富集在有丝分裂G2/M转变等细胞周期相关的生物学过程,Ⅲ期ESCC患者差异表达circRNA的宿主基因主要富集在有丝分裂纺锤体检查点和细胞基质黏附等细胞分裂和肿瘤发展相关的生物学过程。KEGG富集分析显示,Ⅰ期和Ⅲ期ESCC患者的癌组织中差异表达的circRNA均主要富集在细胞黏附相关肿瘤生物学通路。维恩分析结果显示,Ⅰ期和Ⅲ期ESCC患者中筛选出在癌旁组织中特异性表达且差异显著的circRNA分别有2个和8个,在癌组织中特异性表达且差异显著的circRNA分别有11个和14个。circRNA-miRNAmRNA网络显示,Ⅰ期ESCC患者癌组织相关circRNA-miRNA-mRNA网络由7个circRNA节点、10个miRNA节点和28个mRNA节点组成,Ⅲ期ESCC患者癌组织相关circRNA-miRNA-mRNA网络由7个circRNA节点、9个miRNA节点和49个mRNA节点组成。分别选取Ⅰ期和Ⅲ期ESCC患者癌组织中差异表达最显著的hsa-circ-0060927和hsa-circ-0109301进行细胞学和组织学验证,结果显示hsa-circ-0060927在ESCC细胞株TE1、TE13、KYSE30、KYSE170和人正常食管上皮细胞株HEEC的相对表达量分别为7.82±1.96、12.69±2.68、12.78±2.74、7.53±1.75、2.43±0.17,差异有统计学意义(F=4.68,P=0.004),hsa-circ-0060927在ESCC细胞株TE1、TE13、KYSE30、KYSE170中的相对表达量均高于人正常食管上皮细胞株HEEC,差异均有统计学意义(P=0.009;P=0.003;P=0.003;P=0.007)。hsa-circ-0109301在ESCC细胞株TE1、TE13、KYSE30、KYSE170和人正常食管上皮细胞株HEEC中的相对表达量分别为5.16±1.32、6.28±1.57、4.89±1.13、8.92±2.12、22.56±4.13,差异有统计学意义(F=4.31,P=0.022),hsa-circ-0109301在ESCC细胞株TE1、TE13、KYSE30、KYSE170中的相对表达量均低于人正常食管上皮细胞株HEEC,差异均有统计学意义(P=0.027;P=0.015;P=0.024;P=0.008)。hsa-circ-0060927在13例早期ESCC患者癌组织中的相对表达量为12.89±2.67,明显高于癌旁组织的5.73±1.18,差异有统计学意义(t=15.02,P<0.001);hsa-circ-0109301在19例晚期ESCC患者癌组织中的相对表达量为7.78±2.17,明显低于癌旁组织的16.32±3.15,差异有统计学意义(t=9.73,P<0.001)。hsa-circ-0109301的表达与晚期ESCC患者分化程度相关(P=0.023)。结论在早期和晚期ESCC患者中分别筛选出1个特异性差异表达最显著的circRNA(hsa-circ-0060927和hsa-circ-0109301),其中hsa-circ-0060927在ESCC癌组织中高表达,hsa-circ-0109301在ESCC癌组织中低表达,且hsa-circ-0109301的表达与肿瘤分化程度相关。

Objective To analyze circRNAs specifically differentially expressed in esophageal squamous cell carcinoma(ESCC)based on high-throughput sequencing data.Methods Six patients with pathologically confirmed ESCC in Tangdu Hospital of Air Force Medical University from March 2018 to March 2019 were selected as the research subjects,among which 3 were stageⅠESCC and 3 were stageⅢESCC.Highthroughput sequencing technology was used to analyze the difference in the expression of circRNA in cancer tissues and adjacent tissues of patients.GO enrichment analysis,KEGG enrichment analysis and Venn analysis were performed on differentially expressed genes.The circRNA-miRNA-mRNA network was constructed using Cytoscape software.The most significantly differentially expressed genes in cancer tissues were verified in cells and tissues,and the relationships between circRNAs and clinical pathological indicators of patients were analyzed.Results A total of 553 differentially expressed circRNAs were screened in paracancerous tissues and cancer tissues of 3 stageⅠESCC patients,of which 413 were up-regulated and 140 were down-regulated in cancer tissues;A total of 425 differentially expressed circRNAs were screened in paracancerous tissues and cancer tissues of 3 stageⅢESCC patients,of which 276 were up-regulated and 149 were down-regulated in cancer tissues.GO enrichment analysis showed that the host genes of differential circRNAs in patients with stageⅠESCC were mainly enriched in cell cycle-related biological processes such as mitotic G2/M transition.The host genes of differential circRNAs in patients with stageⅢESCC were mainly enriched in biological processes related to cell division and tumor development,such as mitotic spindle checkpoint and cell matrix adhesion.KEGG enrichment analysis showed that the differential circRNAs in cancer tissues of stageⅠand stageⅢESCC patients were mainly enriched in cancer-related biological pathways such as cell adhesion.The results of Venn analysis showed that in stageⅠESCC patients and stageⅢESCC patients,2 and 8 circRNAs that were only specifically expressed in paracancerous tissues and had significant differences were screened out respectively,and were only specifically expressed in cancer tissues with significant differences were 11 and 14 respectively.The circRNA-miRNA-mRNA network showed that the cancer tissuerelated circRNA-miRNA-mRNA network in stageⅠESCC patients consisted of 7 circRNA nodes,10 miRNA nodes and 28 mRNA nodes,and the cancer tissue-related circRNA-miRNA-mRNA network in stageⅢESCC patients consisted of 7 circRNA nodes,9 miRNA nodes and 49 mRNA nodes.The most significantly differentially expressed hsa-circ-0060927 and hsa-circ-0109301 in cancer tissues of patients with stageⅠESCC and stageⅢESCC were selected for cytological and histological verification.The results showed that the relative expression levels of hsa-circ-0060927 in ESCC cell lines TE1,TE13,KYSE30,KYSE170,and human normal esophageal epithelial cell line HEEC were 7.82±1.96,12.69±2.68,12.78±2.74,7.53±1.75,and 2.43±0.17,respectively,with a statistically significant difference(F=4.68,P=0.004).The relative expression levels of hsa-circ-0060927 in ESCC cell lines TE1,TE13,KYSE30,and KYSE170 were higher than that in human normal esophageal epithelial cell line HEEC,with statistically significant differences(P=0.009;P=0.003;P=0.003;P=0.007).The relative expression levels of hsa-circ-0109301 in ESCC cell lines TE1,TE13,KYSE30,KYSE170,and human normal esophageal epithelial cell line HEEC were 5.16±1.32,6.28±1.57,4.89±1.13,8.92±2.12,and 22.56±4.13,respectively,with a statistically significant difference(F=4.31,P=0.022).The relative expression levels of hsa-circ-0109301 in ESCC cell lines TE1,TE13,KYSE30,and KYSE170 were lower than that in human normal esophageal epithelial cell line HEEC,with statistically significant differences(P=0.027;P=0.015;P=0.024;P=0.008).The expression level of hsa-circ-0060927 in cancer tissues of 13 early ESCC patients was 12.89±2.67,significantly higher than 5.73±1.18 in paracancerous tissue,and there was a statistically significant difference(t=15.02,P<0.001);the expression level of hsa-circ-0109301 in cancer tissues of 19 patients with advanced ESCC was 7.78±2.17,significantly lower than 16.32±3.15 in paracancerous tissue,and there was a statistically significant difference(t=9.73,P<0.001).The expression of hsa-circ-0109301 was related to the degree of tumor differentiation in advanced ESCC patients(P=0.023).Conclusion One circRNA(hsa-circ-0060927 and hsa-circ-0109301)with the most significanty differential expression is selected in early and advanced ESCC patients respectively,in which hsa-circ-0060927 is highly expressed in ESCC cancer tissues and hsa-circ-0109301 is lowly expressed in ESCC cancer tissues,and the expression of hsa-circ-0109301 is correlated with the degree of tumor differentiation.