CircACTG1 Promotes Hepatocellular Carcinoma Progression By Regulating MiR-940/RIF1 Axis And Activating AKT/mTOR Pathway

Objective:To investigate the expression of circACTG1(hsa＿circ＿0046144)in hepatocellular carcinoma(HCC)and its possible regulation mechanism.It provides a new strategy for molecular detection and treatment of hepatocellular carcinoma.Methods:By downloading the public database GEO(GSE77314),bioinformatics methods were used to analyze and screen out circ RNA with differential expression in liver cancer,and select genes with high differential expression.Twenty cases of hepatocellular carcinoma and 20 matched paracancer tissues were collected from the Department of Hepatobiliary Surgery,Affiliated Hospital of Guizhou Medical University.The expression of circ ACTG1 was detected by quantitative polymerase chain reaction(q PCR).Subsequently,the expression of circ ACTG1 in HCC cell lines was detected by q PCR.The expression of circ ACTG1 in cancer and adjacent tissues was detected by in situ hybridization(ISH).Clinical data of 20 patients were collected for receiver operating characteristic(ROC)curve to evaluate the diagnostic value of circ ACTG1 for disease.Lentivirus was used to construct stable knockdown circ ACTG1 cell line,and q PCR was used to detect its expression in HCC cell lines.CCK8 was used to detect the changes of cell proliferation after knockdown of circ ACTG1.The change of cell migration ability after knockdown circ ACTG1 was detected by scratch test.Transwell assay was used to detect the changes of invasion ability of circ ACTG1 cells after knockdown.Subsequently,circ Base database was used to predict the downstream regulatory mechanism of circ ACTG1,and the mi RNA with potential interaction was predicted to be mi R-940.Double luciferase reporter gene assay and q PCR were used to verify the relationship between circ ACTG1 and mi R-940.Spearman correlation coefficient was used to analyze the correlation between circ ACTG1 and mi R-940.After co-transfection of circ ACTG1 with mi R-940 inhibitor,q PCR was used to detect the expression of mi R-940 inhibitor.CCK8,cloning formation and Transwell were used to detect the changes of cell proliferation and invasion ability.Target gene RIF1 was predicted using mi RNA target gene database Target Scan.The cells were divided into three groups: mi R-940 overexpression group,circ ACTG1 interference group and mi R-940 interference +circ ACTG1 interference group.The expression of RIF1 was detected by q PCR and Western Blot respectively.The overall survival(OS)of RIF1 was analyzed using the Cancer Genome Atlas(TCGA)database.Western Blot assay was used to detect the changes of AKT-m TOR pathway proteins in the three groups.The effect of circ ACTG1 on the proliferation of hepatocellular carcinoma cells was verified in vivo(subcutaneous tumor-forming experiment in nude mice).Results:The circRNA with the highest differential expression ratio in liver cancer was selected as circ ACTG1 by GEO chip(GSE77314).The expression of circ ACTG1 in HCC tissues was significantly higher than that in paracancer tissues,and the expression of circ ACTG1 in HCC cell lines was increased,especially in MHCC-97 H and Huh-7 cells.ISH results showed that circ ACTG1 expression was significantly increased in cancer tissues compared with adjacent tissues.OS curve showed that the prognosis of patients with high expression of circ ACTG1 was poor.ROC curve showed that the sensitivity and specificity of circ ACTG1 for the diagnosis of HCC was 78.9% and 76.2%.The proliferation,migration and invasion of circ ACTG1 cells were significantly reduced after knockdown.The prediction of circ Base database found that the downstream regulatory mechanism of circ ACTG1 was related to mi R-940.The results of the dual luciferase report showed that compared with the NC group,the luciferase activity was significantly decreased in the circ ACTG1-wt group co-transfected with mi R-940 mimic,while there was no significant difference in the luciferase activity in the circ ACTG1-mut group.Spearman correlation analysis showed that circ ACTG1 was negatively correlated with mi R-940 expression(r=-0.64).mi R-940 inhibitor could restore the inhibitory effect of shcirc ACTG1 on the proliferation and invasion of HCC cells.The expression level of RIF1 in the mi R-940 overexpression group was significantly reduced,and that in the circ ACTG1 interference group was significantly reduced,and the expression level of RIF1 in the mi R-940 interference + circ ACTG1 interference group was recovered.The Cancer Genome Atlas(TCGA)found that RIF1 with high expression had a better prognosis.The expression of phosphorylated AKT and m TOR declined in mi R-940 overexpression group and circ ACTG1 interference group,the expression of phosphorylated AKT and m TOR increased mi R-940 interference+ circ ACTG1 interference group.In vivo experimental results showed that compared with the control group,the volume and weight of subcutaneous tumor were significantly decreased after circ ACTG1 interference.Tumor volume and weight were significantly increased in the shcirc ACTG1+ Antagomir-940 response group compared with the circ ACTG1 interference group.Conclusions:The expression of circACTG1 is significantly increased in HCC,and it has certain diagnostic value.Moreover,circ ACTG1 potentially regulates HCC cell proliferation,migration and invasion via the miR-940/RIF1 /AKT/mTOR pathway.