Differential Expression And Function Prediction Of Circular RNAs In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC),the fifth most common cancer in the world,ranks the third in cancer-related death.circularRNAs(circRNAs)are a new class of abundant endogenous functional non-codingRNAs,and have been demonstrated to play important roles in tumorigenesis and development of HCC.We aimed to identify differentially expressed circRNAs(DECs)from circRNA expression data of HCC tissues and paired adjacent non-tumor tissues,and analyze their possible binding miRNAs,downstream targets and associated cell signaling pathways,sequence construction,as well as functional enrichment of their parental gene by bioinformatics,to predict possible functions and mechanisms of these DECs in tumorigenesis and development of HCC.CircularRNA expression profiles of HCC tissues and paired adjacent non-tumor tissues GSE94508(five pairs of tissues)and GSE97332(seven pairs of tissues)were downloaded from GEO database,and GEO2 R was used to select DECs.Several miRNA databases were used to predict DEC-miRNA and miRNA-mRNA interaction,the results of which were combined with those from literatures to determine several essential miRNAs,which could bind with two or more DECs and have been validated to play pivotal roles in HCC progression by targeting multiple genes.For essential miRNAs,DAVID,Panther and STRING were used to perform KEGG and Panther enrichment analysis,as well as protein-protein interaction(PPI)analysis,and Cytoscape was used to visualize essential miRNAs-centered circRNA-miRNA-mRNA regulatory network.circ Base,UCSC and NCBI database were used to determine sequence construction information of DECs.DAVID and Panther were applied to perform GO,KEGG,and Panther enrichment analysis of parental genes of DECs.Finally,several DECs were randomly selected,and their differnental expression in HCC cells was confirmed by quantitative real-time PCR(q RT-PCR)and Sanger sequencing.87 circRNAs were identified as DECs(76 up-regulated,11 down-regulated)between HCC tissues and paired adjacent non-tumor tissues.By prediction of DEC-miRNA and miRNA-mRNA interaction combined with results from literatures,four essential miRNAs,which could bind with two or more DECs and have been validated to play pivotal roles in HCC progression by targeting multiple genes,including hsa-miR-7-5p,hsa-miR-145-5p,hsa-miR-203a-3p and hsa-miR-192-5p.Essential miRNA-centered circRNA-miRNA-mRNA regulatory network was constructed,and many genes of which enriched in cancer-associated pathways,such as Wnt,TGF-β and Ras.The sequence construction of majority DECs were exon type.Parental genes of DECs were enriched in 28 GO items,two KEGG and six Panther pathways.Among nine randomly selected DECs,seven were detectable,while two DECs,including hsa＿circ＿0001489 and hsa＿circ＿0039053,were undetectable in both human normal liver cell line L02 and human HCC cell line Hep G2.Among seven detectable DECs,compared with L02,expression of six DECs,including hsa＿circ＿0001806,hsa＿circ＿0003528,hsa＿circ＿0008583,hsa＿circ＿0009910,hsa＿circ＿0032704 and hsa＿circ＿0065214,were increased in Hep G2 cells,whereas expression of hsa＿circ＿0007762 was reduced in Hep G2 cells;the expression pattern of which were consistent with those analysis results from circRNA array data in GEO database.q RT-PCR products of these seven DECs were with high specificity.Many circRNAs were differentially expressed in HCC,which may be closely correlated with tumorigenesis and progression of HCC.circRNA-miRNA-mRNA regulatory network suggested that complicated regulatory relationships existed among circRNA,miRNA and mRNA.By targeting multiple miRNAs,DECs may regulate expression of various downstream genes of these miRNAs,as well as associated signaling pathways,and thus impacting on tumorigenesis and progression of HCC.The majority of DECs were exon type,indicating that the majority of DECs may locate at cytoplasm,and function as miRNA sponge.Parental genes of DECs enriched in many cancer-related biological processes and pathways,and DECs may involve in HCC tumorigenesis and progresssion by modulating transcription of their parental genes.Finally,differential expression of seven DECs was further confirmed at cell levels,including hsa＿circ＿0001806,hsa＿circ＿0003528,hsa＿circ＿0008583,hsa＿circ＿0009910,hsa＿circ＿0032704and hsa＿circ＿0065214,suggesting that these DECs may serve as biomarkers for early diagnosis and prognosis of HCC,or may impact on tumorigenesis and progression of HCC by modulating biological functions of hepatocytes via binding to specific miRNAs or proteins.