Circular RNA circZNF566 Promotes Hepatocellular Carcinoma Progression by Sponging miR-4738-3p and Regulating TDO2 Expression

Abstract

Background: As a recently discovered noncoding RNA, circular RNAs (circRNAs) have been identified to play key roles in cancer biology; however, the detailed functions and mechanisms of circRNAs in hepatocellular carcinoma (HCC) remain largely unclarified. Methods: RNA-seq analysis was used to screen the expression profiles of circRNAs in HCC. CircZNF566 expression in HCC tissues and cell lines was detected by qRT-PCR. In vitro CCK-8, colony formation, wound healing, transwell migration and invasion assays and in vivo tumorigenesis and metastasis assays were conducted to determine the functions of circZNF566. Luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were also performed to confirm the relationship between circZNF566 and miR-4738-3p. Bioinformatics analysis and luciferase reporter assays were employed to determine whether miR-4738-3p regulates tryptophan 2,3-dioxygenase (TDO2) expression. Finally, immunohistochemistry (IHC) was used to detect the level of TDO2 and determine its prognostic value. Results: CircZNF566 was significantly upregulated in HCC tissues and cell lines. High circZNF566 expression in HCC tissues was positively correlated with clinicopathological features and poor prognosis. Functionally, in vitro experiments showed that circZNF566 promoted HCC cell migration, invasion and proliferation, whereas in vivo experiments showed that circZNF566 promoted tumorigenesis and metastasis. Mechanistically, circZNF566 acted as a miR-4738-3p sponge to relieve the repressive effect of miR-4738-3p on its target TDO2. In addition, miR-4738-3p suppressed HCC cell migration, invasion and proliferation, while TDO2 was positively correlated with pathological features and poor prognosis and promoted cell migration, invasion and proliferation in HCC. Conclusion: CircZNF566 is a novel tumor promoter in HCC and functions through the circZNF566/ miR-4738-3p /TDO2 axis; in addition, circZNF566 may serve as a novel diagnostic marker, prognostic indicator and target for the treatment of HCC. Funding Statement: This study was supported by funding from the National Natural Science Foundation of China (No.81670595) and (No. 8197032698), and the Scientific Research Planning Project of Science and Technology Commission of Shanghai (No.15411962700). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Ethics approval was granted by the Ethics Committee of Shanghai General Hospital. Animal experiments were carried out according to the Shanghai General Hospital Animal Care and Use Guidelines, and the experimental protocols were approved by the Shanghai Resource Center of Laboratory Animals of the Chinese Academy of Science. Written informed consents were obtained from all patients.