The Effects Of CYP2E1 Gene On Hepatoma Cell Proliferation,Migration And Invasion Through MAPK-and PI3K-AKT/HDAC6 Signaling Pathway

Background and Objective:Primary Liver Carcinoma(PLC)is the fourth leading cause of cancer and the second cause of human death in malignant tumors.The molecular mechanisms of HCC development have not been systematically elucidated.Although the diagnosis and treatment strategies of HCC have been improving,the survival rate is still low due to the complex pathogenesis,metastasis,insensitivity to chemotherapy and recurrence after surgical resection.Therefore,it is of great practical significance to conduct further research on the molecular mechanisms of occurrence and progression of HCC to find new therapy.Cytochrome P450 oxidase(CYP)2E1 is a major enzyme which plays a vital role in drug-induced hepatotoxicity mainly involved in the metabolism of pro-carcinogen and environmental toxicants.Previous studies have mainly focused on the role of CYP2E1 in activating pro-carcinogens in vivo such as ethanol,nitrosamines,generating carcinogens,resulting in gene mutation and inducing cancer.However,it was reported that the expression of CYP2E1 in HCC tissues gradually decreased accompanied with its progression and lower expression of CYP2E1,which was associated with the malignant characteristics and shorter survival.The specific biological role and mechanism of the progression of HCC are still largely uncertain.In this study,CYP2E1 was detected in different liver cancer cell lines and tissues to explore the correlation between the concentration of CYP2E1 and the survival time of HCC patients.Next,in vitro and in vivo,CYP2E1 were determined in SK-Hepl and HCC-LM3 cells to evaluate the p38MAPK and PI3K-Akt/p-HDAC6 signaling pathway on cell proliferation,migration and invasion of HCC.CYP2E1 would probably be a new biomarker for HCC or a new gene and molecular target for the management of HCC.Method:1.Examination of the significance of CYP2E1 in different liver cancer cell lines and tissues1)Download HCC data from TCGA(Cancer Genome Atlas)database for bioinformatics analysis to explore the significance of CYP2E1 in the occurrence,development and prognosis of HCC.2)Real-time PCR was used to determine the differences of CYP2E1 gene expressions between normal live cell lines(LO2)and HCC cell liens(SK-Hep1,HepG2,HCC-LM3 and SMMC-7721).3)HE staining and immunohistochemical(IHC)staining were applied to observe the pathological morphology and to evaluate expression of CYP2E1 protein in 44 cases of HCC and adj acent tissues.2.Investigation of the significance of CYP2E1 expression in SK-Hep1 and HCC-LM3 cells1)The coding sequence of CYP2E1 was cloned into the lentiviral vector pCDH-EGFP in vitro,and pCDH-EGFP-CYP2E1 lentiviral vector was constructed,which was transfected into SK-Hep1 and HCC-LM3 cells to construct lentivirus-mediated,stably transduced overexpressing CYP2E1 Cell Line.2)The impact of CYP2E1 on SK-Hep1 and HCC-LM3 cell proliferation was evaluated by CCK8,Plate clone formation and Edu assays.3)Scratch-wound assay and Transwell assay were conducted to evaluate the effect of CYP2E1 on SK-Hep1 and HCC-LM3 cell migration and invasion.4)Flow cytometry was subjected to evaluate the effects of CYP2E1 overexpression on the cell cycle phase distribution of SK-Hep1 and HCC-LM3.5)Western blot assay was performed to detect proliferation-related proteins and antigens in SK-Hep1 and HCC-LM3 cells.6)A contracted nude mouse model which was made by the subcutaneous injection of xenograft tumor and lung metastasis tumor was subjected to study the effect of CYP2E1 on SK-Hep1 and HCC-LM3 cells-mediated tumorigenic ability,invasion and metastasis ability.7)IHC staining was used to observe the expression of proliferation-related antigen Ki67 in subcutaneous tumors of nude mice.3.Exploring molecular mechanism of CYP2E1-mediated HCC cell proliferation,invasion and migration1)At first,we use TCGA database to evaluate the difference of HDAC6 gene expression between cancer and paraneoplastic tissues as well as the patterns of CYP2E1 and HDAC6 gene expression.Second,following the overexpression of CYP2E1,we detected HDAC6 mRNA expression.2)Following the overexpression of CYP2E1 in SK-Hep1 and HCC-LM3 cells,the cells were subjected to evaluate HDAC6 expression,cell proliferation ability and proliferation-related proteins.3)Following the silencing of CYP2E1 in SK-Hep1 and HCC-LM3 cells,the cells were subjected to evaluate HDAC6 expression,cell proliferation ability and proliferation-related proteins.4)Following the overexpression of CYP2E1 in SK-Hep1 and HCC-LM3 cells,the cells were subjected to evaluate cell proliferation ability using Edu kit in presence,absence of HDAC6 inhibitor tubastatin and proliferation-related proteins.5)We have performed p-HDAC6 protein expression in cancer and paraneoplastic tissues by IHC staining using its antibody.Result:Chapter 1:1)In the TCGA database,we found the expression level of CYP2E1 in the cancer tissue was significantly lower than that of the paraneoplastic tissues[median-2.586(-3.661--1.649),P<0.001].Kaplan-Meier survival analysis showed that the overall survival period of patients with low CYP2E1 expression was significantly shorter than that of patients with high CYP2E1 expression(P=0.03).2)Compared with normal hepatocyte LO2 cell line,CYP2E1 mRNA expression was down-regulated in the SK-Hep1,HepG2,HCC-LM3,and SMMC-7721 cells,and SK-Hep1 and HCC-LM3 cells had the lowest expression than that of others(P<0.01).3)IHC staining results showed that the CYP2E1 expression of the cancer tissues was lower than paraneoplastic tissues.Chapter 2:1)The data of the CCK8 Plate clone formation and Edu assays demonstrated that CYP2E1 overexpression impaired cell proliferation ability in SK-Hep1 and HCC-LM3 cells.2)Scratch-wound and Transwell assays revealed that overexpression of CYP2E1 inhibited SK-Hep1 cell migration.3)Flow cytometry revealed that CYP2E1 induced SK-Hep1 and HCC-LM3 cells arrested in G1 phase;the quantitative data of Western Blot analysis showed that the expression of Cyclin D1,CD4,CD6 and Ki67 was significantly reduced in the overexpressed CYP2E1 of SK-Hep1 and HCC-LM3 cells.4)In vivo data of the nude mice showed that the tumor volumes of the mice injected CYP2E1 overexpressed SK-Hep1 and HCC-LM3 cells were significantly smaller than that of the control group.5)The results of HE staining showed that the lung tissue of nude mice received the tail vein injection of the CYP2E1 low expression liver cancer cells had obvious lung metastases but no lung metastases in mice receiving CYP2E1 overexpressed cancer cells.6)Similarly IHC staining results showed that the Ki67 positive cells of the tumor tissues of the mice receiving the subcutaneous xenograft tumors of the CYP2E1 overexpression cell was significantly reduced than that of control mice.Chapter 3:1)The data of TCGA database showed that HDAC6 expression was lower in cancer tissue than in the paraneoplastic tissues.The data of transcriptome sequencing and bioinformatics indicated that HDAC6 may involve in CYP2E1-mediated malignant progression of HCC.2)The results of Western Blot showed that the expression of HDAC6 protein was increased in the CYP2E1 overexpressed liver cancer cell line accompanied by the reduction of p-p38MAPK,p-PI3K,and p-Akt protein expression levels.3)CYP2E1 silencing decreased the expression levels of p-HDAC6 protein but it increased the expression levels of p-p38MAPK,p-PI3K,and p-Akt proteins.4)The Edu test results showed that the HDAC6 inhibitor tubastatin A enhanced proliferation ability of the liver cancer cell lines with overexpressed CYP2E1.5)IHC staining results showed that the expression of p-HDAC6 was decreased in HCC tissues but increased in normal paraneoplastic tissues.Conclusion1.The expression level of CYP2E1 protein in liver cancer tissues is correlated with patient survival period positively.2.Overexpression of CYP2E1 inhibits the proliferation,migration and invasion of liver cancer cell lines.3.CYP2E1 can promote the phosphorylation of p-HDAC6 via reduction of p38MAPK-and PI3K/AKT signaling activation,which leads to ultimately inhibition of HCC cell proliferation and tumor growth.