The Study On Biological Function And Molecular Mechanism Of Circular RNA Circ-CCT3 In Hepatocelluar Carcinoma

Background:Primary hepatocellular carcinoma(HCC)is one of the most common malignancies.At present,the molecular mechanism of liver cancer is unclear.Circular RNA(or circ RNA)is a kind of single-stranded RNA.Unlike the well-known linear RNA,it forms a covalently closed continuous loop,that is,in circular RNA,usually found at the 3 ’and 5’ ends of the RNA molecule linked.This function confers a variety of properties to circular RNA.Recently,the imbalance of circular RNA(circ RNA)has been shown to play an important regulatory role in cancer development and progression.However,the role of most circ RNAs in liver cancer remains unknown.Aims:The circular RNA microarray was used to analyze differential circular RNAs in liver cancer tissue and adjacent tissue,and we aimed to find a significant circ RNA in the development and progression of hepatocellular carcinoma,and to explore its specific mechanism in the progression of HCC,and provide novel ideas for the diagnosis and treatment of HCC.Methods:In this study,first,six pairs of liver cancer and adjacent tissue samples were analyzed by circ RNA Microarray.Circular RNAs with high expression in liver cancer tissues were selected for verification,and up-regulated circ RNAs were verified in 24 pairs of liver cancer and adjacent tissue by q PCR.The up-regulated circ RNA was identified as circular RNA by RNase R digestion and q PCR.Kaplan-Meier statistical method was used to analyze the relationship between circ RNA and prognosis of HCC patients.Secondly,seven RBPs that could bind to circ-CCT3 were predicted through the starbase database.RIP experiments were used to verify the RBPs that could bind to circ RNA,and Me RIP experiments were used to verify the m6 A levels of circ RNA.In terms of biological functions,in vitro,CCK8 and clone formation assay were performed to detect the effects of circular RNA on the proliferation of liver cancer cells.Transwell experiments were used to detect invasion and migration.Flow cytometry was used to detect the effects of circ RNA on the cell cycle.In vivo,the effects of circular RNA on the proliferation and migration of liver cancer were examined by subcutaneous xenograft in nude mice and lung metastasis experiments in nude mice,respectively.Finally,in terms of molecular mechanism,circ RNA immunoprecipitation,dual luciferase reporter gene analysis,and fluorescence in situ hybridization were used to evaluate the interaction between circ RNA and miRNA.The miRanda database is used to predict the target genes that miRNA may bind to,and q PCR and dual luciferase reporter assay were used to verify the target genes regulated by miRNA.The target genes are analyzed for prolifeation,invasion and migration and cell cycle of liver cancer cells by CCK8、clone formation,transwell and flow cytometry.Results:1.Circ RNA microarray analyzed that 315 circular RNAs were up-regulated in HCC tissues.Circular RNAs hsa＿circ＿0004680 and hsa＿circ＿00014717 are highly expressed in liver cancer tissues.QPCR analyzed the two circ RNAs in 24 pairs of cancer and adjacent tissues,results showed that hsa＿circ＿00014717 has no significant difference between cancer and adjacent tissues,while hsa＿circ＿0004680 was highly expressed in liver cancer tissues,and we named hsa＿circ＿0004680 as circ-CCT3.2.Circ-CCT3 can tolerate RNase R digestion.Nucleus and cytoplasmic separation and FISH assay showed that circ-CCT3 is mainly localized in the cytoplasm.RNA in situ hybridization revealed that circ-CCT3 was highly expressed in HCC tissues.Kaplan-meier curve indicated that up-regulation of circ-CCT3 in HCC was associated with poor prognosis.3.Through the starbase database analysis,we searched out the RNA-binding proteins of circ-CCT3,and select 7 RBPs related to RNA m6 A modification.It was found that knocking down the m6 A demethylase ALKBH5(alk B homolog 5,ALKBH5)can down-regulate the level of circ-CCT3 and knocking down the m6 A methylase METTL3(methyltransferase-like3,METTL3)can up-regulate the level of circ-CCT3.Me RIP experiment shows that ALKBH5 and METTL3 can affect the m6 A modification of circ-CCT3.RIP experiments show that m6 A “reader” protein YTHDF2 can bind to circ-CCT3,and interference with YTHDF2 can up-regulate the level of circ-CCT3.The biological function of circ-CCT3 in hepatocellular carcinoma,in vivo and in vitro data indicate that knockout of circ-CCT3 inhibits proliferation,invasion and migration of HCC cells and angiogenesis.4.Mi Randa database predicted the five miRNAs that circ-CCT3 may bind.It was found by circ RIP assay that circ-CCT3 may bind to miR-378a-3p.The interaction between circ-CCT3 and miR-378a-3p was verified by a dual fluorescence report.FISH experiments confirmed colocalization between circ-CCT3 and miR-378a-3p.5.Predicting the target genes that miR-378a-3p may bind by miRanda database,the target gene with the score >=85 were selected,and we find that miR-378a-3p can decrease the level of FLT1 by q PCR.Dual luciferase report assay confirmed that miR-378a-3p can bind to FLT1.At the same time,over-expression and knock down of circ-CCT3 up-regulated and decreased FLT1 at the RNA and protein levels.Rescue experiments indicated that circ-CCT3 acts as the sponge of miR-378a-3p to regulate FLT1 expression.6.To explore the biological roles of FLT-1 in hepatocellular carcinoma,we found that knockdown of FLT1 can inhibit proliferation,invasion and migration,and angiogenesis of liver cancer cells.Conclusions:1.The expression of circ-CCT3 is increased in liver cancer tissues compared with adjacent tissues,and the high expression of circ-CCT3 is related to the poor prognosis of HCC patients.2.In vitro,knocking down circ-CCT3 can inhibit the proliferation,invasion and migration of HCC cells.In nude mouse animal experiments,interferencing with circ-CCT3 can inhibit the growth of subcutaneous tumors in nude mice and reduce the ability of lung metastasis.3.Circ-CCT3 was modified by m6 A,RNA m6 A demethylase ALKBH5,m6 A methylase METTL3,and m6 A methylation recognition protein YTHDF2 are involved in regulating the expression of circ-CCT3.4.Mechanically,circ-CCT3 can promote the proliferation,invasion and migration of hepatocellular carcinoma through the miR-378a-3p/FLT1 signal axis.