| Objective: Hepatocellular carcinoma(HCC)is one of the most common malignant tumors worldwide which is associated with early metastasis and poor prognosis.From a global perspective,between 75 and 80% of patients with liver cancer have a history of chronic hepatitis B virus(HBV)and hepatitis C virus(HCV)infections.Sub-Saharan Africa and East Asia are regions with a high incidence of hepatocellular carcinoma,and the HCC patients in China account for 50% of the total number of patients around the world,a fact that is inseparable from the huge numbers of people with hepatitis infections in this region.Biological behaviors,such as early invasion,metastasis and recurrence,are challenges to the clinical treatment of liver cancer.Specifically,epithelial-mesenchymal transition(EMT)is considered to be a key step for tumor invasion and metastasis.Tumor cells develop powerful invasive and metastatic ability through the EMT process,which allows the migration of tumor cells to different tissues and organs.At the same time,the X protein(HBx)expressed by the X gene of HBV is mainly involved in the invasion and metastasis of HCC by promoting the process of epithelial-mesenchymal transition.In order to study the mechanism of HBx promoting epithelial-mesenchymal transition of hepatocellular carcinoma,our project intends to determine whether HBx can promote the epithelial-mesenchymal transition of hepatocellular carcinoma through the competing endogenous RNAs(ceRNA)of circRNA/miRNA/mRNA,then identify the circ RNA which can play a key role,and study the specific mechanism of promoting tumor invasion and metastasis.Methods: Firstly,HepG2 cell line was selected as the main cell in our research,then we construct HBx overexpression stable cell line by lentivirus transfection,and the changes of cell phenotype and EMT related protein indexes such as E-cadherin,Ncadherin and vimentin were observed to verify whether HBx overexpression cells have the process of epithelial-mesenchymal transition,and the changes of invasion ability of overexpression cells were verified by Transwell invasion experiment.Then,the HBx overexpression cells and the control cells were sequenced by high-throughput whole transcriptome sequencing,and the circRNA with significant difference in expression was screened,and the miRNA and mRNA that might be combined with it.The sequencing results were further verified by qPCR.After identifying circCYP24A1 as a candidate circrna,we used qPCR in 40 clinical samples to verify whether there was a difference between the expression of circCYP24A1 in liver cancer and adjacent tissues,then,we HBx transfected overexpression cell lines were with lentivirus,knocked down the expression of circCYP24A1,and observed the process of epithelial-mesenchymal transition,including the changes of cell phenotype,EMT related protein expression and cell invasion function.Then,we overexpressed circCYP24A1 in HepG2 cells,and observed whether the process of epithelial-mesenchymal transition happened again,so as to identify the role of circCYP24A1 in the HBx mediated EMT of liver hepatocellular carcinoma.Finally,we determined the qPCR products of circCYP24A1 for TA cloning sequencing to verify whether circCYP24A1 has circle characteristics,and located circ CYP24A1 by fluorescence in situ hybridization(FISH)to observe whether it can be expressed in the cytoplasm.Finally,we further determined whether circCYP24A1 could participate in tumor invasion and metastasis by combining with mi R-506 to form ceRNA mechanism by double luciferase reporter gene assay.Results: After HBx overexpression by lentivirus in HepG2 cells,the expression of Ecadherin decreased,the expression of N-cadherin and vimentin increased,the cell phenotype tended to interstitial phenotype,and the invasion function increased(P<0.05).The expression of circCYP24A1 in the HBx overexpression group was significantly higher than that in the control group,and the predicted combined miRNA-506 expressed in overexpression group was lower than that in the control group,and the expression of Snail2 expressed in overexpression group was correspondingly higher than that in the control group(P<0.05),which was consistent with the sequencing results.Then,qPCR analysis of 40 clinical samples showed that the expression of circCYP24A1 in HCC tissues was significantly higher than that in adjacent tissues(P<0.05),and after lentivirus knockdown of circCYP24A1,the EMT process of HBx overexpression cells was inhibited and the invasion function was decreased(P<0.05).After overexpression of circ CYP24A1 in HepG2 cells,epithelial-mesenchymal transition occurred again.TA cloning sequencing of qPCR products of circCYP24A1,we verified the circle characteristics of circCYP24A1.At the same time,FISH also indicated that circ CYP24A1 was mainly expressed in the cytoplasm.Moreover,the double luciferase reporter gene assay confirmed that cirCYP24A1 could specifically bind to miR-506,inhibit the effect of miR-506 and promote the epithelial-mesenchymal transition of HCC through the action of molecular sponge.Conclusion: Above all,the results reveal that HBx can inhibit the epithelialmesenchymal transition of HCC by competitive inhibition of miR-506 with high expression of circCYP24A1,thus promoting the epithelial-mesenchymal transition of HCC and enhance the invasion and metastasis of HCC. |
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