Screening For And Functional Analysis Of Epigenetic Biomarkers Of CircRNAs In Esophageal Squamous Cell Carcinoma

Background and objectives:Esophageal cancer is a common malignant tumor of the digestive tract and an important health-related problem in many developing countries.China is one of the countries with the highest incidence and mortality of esophageal cancer,with esophageal squamous cell carcinoma?ESCC?as the main subtype.Although progress in the diagnosis and treatment of esophageal cancer have been made in recent years,the prognosis is still poor and the 5-years survival rate is less than 30%,mainly because many patients are diagnosed at an advanced stage.Esophageal cancer in the early stage has no obvious clinical symptoms and lack of effective and non-invasive methods for the early diagnosis.Therefore,searching for sensitive,specific and easily measured biomarkers is useful for the early detection and early treatment of esophageal cancer and improving the prognosis of patients.Circular RNA?circ RNA?is a large class of endogenous non-coding RNA?nc RNA?with regulatory ability.The closed loop structure formed by reverse splicing makes it resistant to RNA exonuclease.Circ RNA has the advantages of extracellular stability and evolutional conservation,and plays a key role in gene regulation.Studies have shown the potential of circ RNA as a biomarker for the diagnosis or prognosis of human diseases.However,to date,few studies have been performed to explore the role of circ RNA in the development and progression of esophageal cancer.Methods: We adopted a three-stage design.In the first stage,we used a high-throughput microarray chip to screen for differentially expressed circ RNAs in 7 pairs of ESCC tissues and adjacent normal-appearing tissues.The criteria to distinguish the different expression between two groups were fold change?FC?of expression ?2 and P <0.05.We drew the heat map and defined the differentially expressed circ RNAs.The candidate circ RNAs were selected for the second stage for validation.In the second stage,we collected 69 pairs of ESCC tissues and adjacent normal-appearing tissues,44 ESCC blood samples and 44 control blood samples from healthy volunteers.The expression was detected by using quantitative reverse transcription polymerase chain reaction?q RT-PCR?.The receiver operator characteristic?ROC?curve and the area under the curve?AUC?were obtained to assess the diagnostic value of the candidate circ RNA in ESCC.In the third stage,we used two types of ESCC cell lines?TE-1 and KYSE-150?and one normal human esophageal epithelial cell line?HET-1A?to investigate the expression of the candidate circ RNA in different cell lines.The overexpression vector of the candidate circ RNA was constructed and transfected into ESCC cell lines TE-1 and KYSE-150.We observed the effect of the candidate circ RNA on cell proliferation,invasion,migration and apoptosis.Bioinformatics analyses were used to predict the potential binding mi RNAs of the candidate circ RNA and was further validated by using the Dual-Luciferase reporter assay.Results: In the first stage,we observed 744 differentially expressed circ RNAs according to the criteria of FC ?2.0 and P <0.05,including 469 up-regulated circ RNAs and 275 down-regulated circ RNAs.Hsa_circ₀030162 was ranked as the top down-regulated circ RNA?FC=12.1?.Thus,in the second stage we selected this circ RNA for validation using the q RT-PCR in a large sample size.The results show that the expression of hsa_circ₀030162 was significantly declined in cancerous tissues as compared with the adjacent normal-appearing tissues?t=4.365,P<0.001?.The expression of hsa_circ₀030162 was also down-regulated in the plasma of patients with ESCC as compared with healthy controls?t=3.940,P<0.001?.If using tissue hsa_circ₀030162 as the diagnostic biomarker,the AUC was 0.740?95% CI: 0.637-0.843?,with a sensitivity of 73.91% and a specificity of 56.52%.If using plasma hsa_circ₀030162 as the diagnostic biomarker,the AUC was 0.736?95% CI: 0.632-0.839?,with a sensitivity of 75.00% and a specificity of 61.36%.When the tissue and plasma hsa_circ₀030162 were combined,the sensitivity and specificity were 81.81% and 65.91%,respectively the AUC was 0.789?95% CI: 0.707-0.890?.The in vitro experiments revealed that hsa_circ₀030162 could promote apoptosis and inhibit proliferation,migration and invasion of ESCC cells.The Dual-Luciferase Reporter assays proved that it could bind to mi R-125a-3p / mi R-218-5p,which are non-coding RNAs involved in carcinogenesis.Conclusions: There was a differential expression profile of circ RNAs in the ESCC tissues and plasma.The expression level of hsa_circ₀030162 was significantly decreased in ESCC.Functional studies have shown that hsa_circ₀030162 plays an important role in regulating cell proliferation,migration,invasion and apoptosis,and has binding sites for cancer-related non-coding RNAs like mi R-125a-3p/mi R-218-5p.The results suggested that circ RNAs,for example hsa_circ₀030162,have a potential value as an epigenetic biomarker in the diagnosis and prognosis of ESCC,which may be a new target for intervention and treatment of esophageal cancer.