Clinical Significance And Biological Mechanism Of Circular RNA Hsa-circ-0046600 In Hepatocellular Carcinoma

Objectives:Circular RNA(circRNA)is critical in the development and progression of various human cancers.Hsa-circ-00446600 is a circular RNA consisting of the anti-shear of exon 4,5,and 6 of its maternal expression gene B3GNTL1,and has a length of 227 bp.However,its function in hepatocellular carcinoma(HCC)is still unclear.The purpose of this study was to investigate the clinical significance,biological function and mechanism of the circular RNA hsa-circ-0046600 in HCC.Methods:The expression level of hsa-circ-0046600 in hepatocellular carcinoma was predicted by analyzing the gene chip GSE97332.Twenty patients with hepatocellular carcinoma from April 2018 to July 2018 in Zhengzhou University People’s Hospital were collected by quantitative real time polymerase chain reaction(qRT-PCR)and fluorescence in situ hybridization(FISH)to detect the expression of hsa-circ-0046600 in HCC tissues and normal tissues,and to analyze the sex,age and clinical pathological tumor size,TNM stage,tumor differentiation and hsa-circ-0046600 by single factor analysis.Whether there is a correlation of vascular invasion.The expression levels of hsa-circ-0046600 in hepatocellular carcinoma-associated cell lines HuH7,HepG2,SMMC-7721,SK-HEP-1,and normal liver cell line HL-7702(L-02)were detected by qRT-PCR.The hsa-circ-0046600 siRNA was constructed to down-regulate the expression of hsa-circ-0046600.Using the scratch test,Western blot was used to detect the migration ability of the siRNA-interfering group and the control group of hepatocellular carcinoma-related cell lines and the expression of the related protein N-cadherin.The miRNAs directly regulated by hsa-circ-0046600 were predicted by bioinformatics,and the luciferase reporter gene assay was used for experimental verification.Meta-analysis was used to confirm the relationship between this miRNA and HCC.Bioinformatics prediction and luciferase reporter gene assays were used to determine hsa-circ-0046600 directly regulated miRNAs,and meta-analysis confirmed the expression of miRNAs in HCC.Bioinformatics prediction of downstream target genes of miRNAs,combined with tumor genome map(The Cancer Genome Atlas,TCGA)to screen target genes of miRNAs in hepatocellular carcinoma;using gene ontology(GO),Kyoto genes and genomes The Encyclopedia of Sciences(KEGG),protein protein interaction network(PPI network),screened miRNA-targeted central mRNA and detected the expression of related proteins by Western blotting.Results:This study showed that the expression level of hsa-circ-0046600 in HCC was significantly higher than that in adjacent normal tissues(P <0.05);it was highly expressed in HCC cell lines,especially HepG2 and SK-HEP-1;hsa-circ-0046600 expression was also associated with tumor size,TNM stage,and pathological vascular invasion.The scratch test showed that the migration ability of HepG2 cells and SK-HEP-1 cells in the transfected hsa-circ-0046600 siRNA group was lower than that in the empty vector group.Bioinformatics shows that the miRNA downstream of hsa-circ-004660 may be miRNA-640.The dual luciferase gene report showed the lowest luciferase activity in hsa-circ-004660WT+hsa-miR-640,indicating hsa-circ-0046600 Can "sponge" to absorb miR-640.Combined with TCGA data,GO analysis,KEGG analysis revealed that HIF-1α may be a downstream protein of the hsa-circ-0046600-miRNA-640 pathway,and HIF-1a in HepG2 and SK-HEP-1 cells after transfection of hsa-circ-0046600 siRNA The expression level was down-regulated compared with the control group,indicating that hsa-circ-0046600 promoted the development of tumor by binding to miR-640 to affect the expression level of HIF-1α and other proteins.Conclusion:1.Circular RNA hsa-circ-RNA0046600 Hepatocellular carcinoma is highly expressed in adjacent tissues.2.In hepatocellular carcinoma,the expression level of hsa-circ-0046600 is related to tumor size,TNM staging and pathological features of pathological vascular invasion;3.Down-regulation of hsa-circ-0046600 expression level,HepG2 and SK-HEP-1 cell migration rate decreased;4.Hsa-circ-0046600 competitively binds to miR-640 and promotes the expression of HIF-1α.