Abstract:

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 stage Ⅰ ESCC and 3 were stage Ⅲ ESCC. High-throughput 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 stage Ⅰ ESCC 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 stage Ⅲ ESCC 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 stage Ⅰ ESCC were mainly enriched in cell cycle-related biological processes such as mitotic G₂/M transition. The host genes of differential circRNAs in patients with stage Ⅲ ESCC 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 stage Ⅰ and stage Ⅲ ESCC patients were mainly enriched in cancer-related biological pathways such as cell adhesion. The results of Venn analysis showed that in stage Ⅰ ESCC patients and stage Ⅲ ESCC 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 tissue-related circRNA-miRNA-mRNA network in stage Ⅰ ESCC patients consisted of 7 circRNA nodes，10 miRNA nodes and 28 mRNA nodes，and the cancer tissue-related circRNA-miRNA-mRNA network in stage Ⅲ ESCC 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 stage Ⅰ ESCC and stage Ⅲ ESCC 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.

Key words: Esophageal squamous cell carcinoma, RNA, circular, Sequence analysis, RNA