Establishment Of Hepatocellular Carcinoma Cell Line HepG2 Stably Over-expressing CYP2E1

Background: Cytochrome P450 participate not only in the metabolic of medicine but also catalysed various precursors of cancer-causing compounds into eventually cancerogen. In addition, our research indicated that CYP2E1 could regulate the gene expression at the level of transcription though metabolism network, such as the classic signal channel(CYP2E1-ROS/RNSM1HM/Nos2). The mechanism mentioned above implicit that the substrate and product of CYP2E1 can play as molecular messenger in signal transduction passway and profoundly affect various cell function. The differential expression of CYP2E1 may play an important role in the before,during and after of tumorigenesis. Especially in liver where the intrinsic expression of CYP2E1 is comparatively high and the expression is easy to induce. The study of CYP2E1 contribute to elucidate the mechanism of liver tumorigenesis and find out the new target of therapy. In this study, we aim at establishing an hepatocellular carcinoma cell line HepG2 which could stably over-expressing CYP2E1 though lentiviral vector. This will lay the foundation for the future series of research.Objective: Acquire complete CYP2E1 gene and construct lentiviral vector Lenti-Cyp2e1-eGFP-Puro. Establish a hepatocellular carcinoma cell line HepG2 which highly expressing CYP2E1. This will create an ideal cell model to study the effect of liver cancer and its molecular mechanism.Methods: Acquire complete CYP2E1 gene though amplify PCR,electrophoretic separation and recycle. Sequencing the CYP2E1 coded sequence by Invitrogen Corporation and compare with coded sequence inUCSC to assure the security and integrate. Construct carrier plasmid Lenti-Cyp2e1-eGFP-Puro.293 T cells were transfected with the recombinant vector, viral supernatant was collected. Infection with lentiviral(Lenti-Cyp2e1-eGFP-Puro) carrying CYP2E1 gene in HepG2 and puromycin-resistance screening were performed to establish the cell line that highly expresses CYP2E1. The transfection conditions was detected through enhanced green fluorescent protein,q-PCR and Western-blotting was used to detect the expression of CYP2E1 in HepG2 and overexpression CYP2E1 cell line。Results: The result of match rate is 100% when compare with CYP2E1 coded sequence which sequencing by Invitrogen Corporation. Transfection though incubate HepG2 with the viral contained culture medium. The cell line of HepG2 express eGFP and emit fluorescence after 12 h. The efficiency of infection between control and experiment group are basic identical. The expression of fluorescent protein increased quickly after 24 h.The expression up to 90% at 4d and approach 100% at 6d. Recover the HepG2-CYP2E1 cell line after 3 months of liquid nitrogen cryopreservation, the expression of fluorescent protein are still close to 100%.The mRNA level of CYP2E1 in HepG2 after infected with lentiviral was 682.70±6.11 times(P < 0.05) than HepG2. There was no statistically significant difference between the HepG2 and vector group for the expression level of mRNA(0.22±0.06 vs 0.36±0.05,P>0.05)and protein(1.26±0.02 vs 1.29±0.01,P>0.05). The protein expression of HepG2 that transfetion of CYP2E1 gene(9.51±0.78)is higher than vecter and HepG2(P < 0.01).Conclusion:1) It is successful to construct an carrier plasmid which could expressCYP2E1.2) Establish an HepG2 cell line overexpression of CYP2E1 stablely by means of lentivector transfection technique.