| Background & Aims:CircularRNA(circRNA)was first discovered existing in RNA viruse.However,due to their low richness in cells,circRNAs were barely getting any attention back then.With the development of the second-generation sequencing technique and bioinformatics,the richness of some circRNAs are found far greater than their originated mRNAs.Now circRNAs are widely known as a novel subset of competing endogenous RNAs,regulating target genes via affecting the function of microRNA and RNA-binding proteins.The role of circRNAs have been studied in various kinds of cancers such as liver cancer,lung cancer,breast cancer,bladder cancer and gastrointestinal cancer.CircularRNA(circRNA)poses great potential in developing pre-clinic diagnosis and refining therapeutic methods in multiple cancers.Yet the underlying correlation between circRNAs and cancer stem cells(CSC)have been poorly understood.Cancer stem cells(CSC)have been proven to take account for the initiation,progression,metastasis,chemo-resistance and relapse of tumors.Hence it is of great urgency to clarify the key circRNAs of liver cancer and its molecular mechanism and discover more effective intervention targets.Methods:We performed circRNA profiling on 10 hepatocellular carcinoma(HCC)samples and paired adjacent normal tissues and circZKSCAN1 which is negatively correlated with CSC markers was secleted as our target circRNA.We used RNA isolation,Rnase R treatment and fluorescence in situ hybridization(FISH)experiments to study the basic biological characteristics of the target circRNA.Using lentivirus system,circZKSCAN1 overexpressing cell lines and their corresponding knockdown ones were constructed,respectively.The sphere formation abilities were analyzed by Spheroid formation assays and In vitro or In vivro limiting dilution assays.RNA-Protein interactions were analyzed by RNA-Binding Protein Immunoprecipitation(RIP)assays.HCC specimen' EpCAM and ki67 protein levels were measured by Immunohistochemistry(IHC).The expression level of circZKSCAN1 was analyzed by in situ hybridization(ISH)staining using a pair of tissue microarrays.Results:High-throughput sequencing showed that circRNAs were differentially expressed in hepatocellular carcinoma and adjacent tissues.CircZKSCAN1 was selected as our target circRNA,which was negatively correlated with the expression of EpCAM.We found that circZKSCAN1 is formed when the 5' splice site at the end of exon 3 is joined to the 3' splice site at the beginning of exon 2.It is resistant to RNA exonucleases and located in the cytoplasm.CircZKSCAN1 suppresses malignant behaviors of hepatocellular carcinoma by downregulating cellular stemness.Moreover,CircZKSCAN1 performs its function through sponging specific RNA-binding protein.In addition,low circZKSCAN1 expression was associated with multiple characteristics of HCC and is of great value to predict the prognosis of patients with liver cancer.Conclusion:In this study,we first used high-throughput sequencing technology to construct circRNAs and mRNA expression profiles of hepatocellular carcinoma tissues,clarified the expression characteristics of circRNAs,and identified the circRNAs related to liver cancer by clinical samples and cluster analysis.circZKSCAN1 was secleted for research.Alu repeat sequences facilitate circZKSCAN1 production,which is resistant to RNA exonucleases and mainly localized in the cytoplasm.Further,we confirmed at the cellular and animal levels that circZKSCAN1 inhibits the expression of stem-related genes such as EpCAM and suppresses malignant behaviors of hepatocellular carcinoma by downregulating cellular stemness.In addition,CircZKSCAN1 performs its function through sponging specific RNAbinding protein FMRP,blocking its binding to other RNAs and inhibiting target gene expression.Our work revealed a whole new theory about how circRNAs regulate malignant behaviors in cancers besides acting as miRNA sponge which we believed offers a new angle for further researches on the relationship between circRNAs and cancers. |
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