Circular RNA profiling and its potential for esophageal squamous cell cancer diagnosis and prognosis

Abstract

Esophageal squamous cell cancer (ESCC) is a high incidence and mortality disease worldwide. However, specificity and sensitivity of its diagnostic and prognostic biomarkers are still unsatisfactory. Recently, circular RNAs (circRNAs) as biomarkers have been studied extensively while the expression profile and clinical significance of circRNAs in ESCC have rarely been studied. We performed circular RNA microarray in 3 pairs of ESCC frozen tumor and non-tumor tissues to identify ESCC-related circRNAs and found 1045 up-regulated and 1032 down-regulated circRNAs among which 6 circRNAs (hsa_circ_0062459, hsa_circ_0076535, hsa_circ_0072215, hsa_circ_0042261, hsa_circ_0001946, and hsa_circ_0043603) displayed consistency with microarray results by qRT-PCR. 3 circRNAs (hsa_circ_0062459, hsa_circ_0001946, and hsa_circ_0043603) were also detected in plasma and 2 of them except hsa_circ_0062459 could be used as diagnostic biomarkers and found in exosome of cell-conditioned culture conditioned media. The AUC, sensitivity and specificity of hsa_circ_0001946 were 0.894, 92, 80%, of hsa_circ_0043603 were 0.836, 64, 92% while a signature combining them were 0.928, 84 and 98%. Hsa_circ_0001946 was confirmed to predict the recurrence, overall survival (OS) and disease-free survival (DFS) in frozen and FFPE tissues, while its overexpression decreased cell proliferation, migration, and invasion.

Keywords: Biomarkers; Circular RNAs; Esophageal squamous cell cancer; Exosome; hsa_circ_0001946.

Fig. 1

Expression pattern of circRNAs by qRT-PCR in plasma and exosome from cell culture conditioned media. a~c The expression levels of 3 circRNAs were detected by qRT-PCR in plasma from 50 ESCC patients and 50 healthy people. Student’s t-test was used for differential analysis and data were presented as the means± SEM. d Receiver operating characteristic (ROC) curve analysis of differentially expressed circRNAs between ESCC patients and healthy controls. The signature was a combination of hsa_circ_0043603 and hsa_circ_0001946. The AUC, sensitivity, and specificity values were given in the graph. AUC: Area Under The Curve; 0043603: hsa_circ_0043603; 0001946: hsa_circ_0001946; signat:signature. e~f The expression levels of hsa_circ_0001946 and hsa_circ_0043603 were detected by qRT-PCR in exosome from cell culture conditioned media of Eca109, Eca9706 and Het-1A. One-way ANOVA was used for differential analysis and data were presented as the means± SEM

Fig. 2

Prognostic significance of hsa_circ_0001946 in ESCC patients. a Kaplan-Meier analysis of disease-free survival (DFS) based on hsa_circ_0001946 expression in 50 frozen tumor tissues of ESCC patients by qRT-PCR. b Kaplan-Meier analysis of DFS based on hsa_circ_0001946 expression in 50 FFPE tissues of ESCC patients by qRT-PCR. c Kaplan-Meier analysis of DFS based on hsa_circ_0001946 expression in 50 frozen tumor tissues and 50 FFPE tissues of ESCC patients by qRT-PCR. d Kaplan-Meier analysis of DFS based on hsa_circ_0001946 expression in 100 FFPE tissues of ESCC patients above by FISH. e Kaplan-Meier analysis of overall survival (OS) based on hsa_circ_0001946 expression in 50 frozen tumor tissues of ESCC patients by qRT-PCR. f Kaplan-Meier analysis of OS based on hsa_circ_0001946 expression in 50 FFPE tissues of ESCC patients by qRT-PCR. g Kaplan-Meier analysis of OS based on hsa_circ_0001946 expression in 50 frozen tumor tissues and 50 FFPE tissues of ESCC patients by qRT-PCR. h Kaplan-Meier analysis of OS based on hsa_circ_0001946 expression in 100 FFPE tissues of ESCC patients above by FISH

Fig. 3

Hsa_circ_0001946 overexpression affects the proliferation, migration, and invasion of Eca109. (a) ESCC cell lines (Eca109, TE-1, K30, and K50) were transfected with lentivirus and the expressions of hsa_circ_0001946 were elevated a lot by qRT-PCR. (b) MTT method showed that hsa_circ_0001946 overexpression inhibited Eca109 proliferation after 48 h. (c) Wound-healing assay indicated that hsa_circ_0001946 overexpression decreased Eca109 migration after 24 h. (d) Transwell assays showed that hsa_circ_0001946 overexpression decreased Eca109 migration and invasion after 48 h incubation. (n = 3; Student’s t-test was used for significance test and data were presented as the means± SEM). (E) Subcutaneous xenografts excised from nude mice. (n = 6;Student’s t-test was used for significance test and data were presented as the means± SEM)