The Activation Of RAS-Raf-MEK-ERK Signaling Pathway By HBx Protein Reversed By CYP3A4 In Hepatoma Cell Lines

Background and ObjectionHepatocellular carcinoma(HCC)is the fifth leading incidence and mortality cancer worldwide.In China,Chronic hepatitis involving in hepatitis B virus(HBV)infection is the major etiology factor for the initiation and development of HCC.Hepatitis B virus x protein(HBx),a HBV-encoded transcriptional co-activator,plays an important role both in virus replication and in hepatocarcinogensis.As a multifunctional regulator,HBx could participate in multiple cell signalling pathways by protein-protein interaction to regulate transcriptional activation,cell cycle,DNA stability and apoptosis.As we all know,liver is the largest metabolic organ in our body,can metabolize all endognous and exogenous substances to maintain the homeostasis in organisms.Drug-metabolizing enzymes,which are responsible for the biotransformation and elimination of endogenous and xenobiotics,have come to be recognized as the first-line defense of liver from potential toxins and carcinogens.Among all of these enzymes,the first-pass of biotransformation was mainly carried out by CYP3A4,the most abundant isoform of monooxygenases,which governs the metabolism of over 50%therapeutic drugs and 30%endogenous substances.Compared to the parent compound,some metabolites of drugs will be more effectiveness,and some will be less toxicity after CYP3A4-mediate biotransformation.Overwhelming evidence demonstrated that dysfunction of CYP3A4 was closely related to the pathology of many liver diseases such as liver cancer,non-alcoholic fatty liver disease,hepatic fibrosis,cirrhosis,etc.Therefore,the expression and functional activity of drug metabolizing enzymes are directly related to the normal physiological function and health status of our body.Many studies have shown that when we suffered from hepatic diseases,such as non-alcoholic fatty liver disease(NAFLD)and hepatic cirrhosis,the function of drug-metabolizing enzymes would reduce.The influence of gene transcription could decrease the expression of drug-metabolizing enzymes.If the function of drug-metabolizing enzyme has been impaired,the ability to remove the harmful substances will reduce,and further damage to the body,promote the development of the disease process.As everyone knows that,the expression of drug metabolizing enzymes is suppressed in HCC.But the interaction among the drug-metabolizing enzymes,HBx,HCC was unclear.The lowering function of drug metabolism just because of HCC or one inducement of which was unknown.If we make the interaction among drug-metabolizing enzymes,HBx and HCC clear,we may find out a new target to the treatment of HCC.MethodsClinical tumor samples in this study were selected from HCC patients with HBV infection background.By using gene chip technology to screen out the drug metabolizing enzymes which correlate the most with HBx protein in HCC tissues.In the end,CYP3A4 and RAS-Raf-MEK-ERK signaling pathway have the highest expression and relevance in clinical HCC cases.For ascertaining the relationship among CYP3A4,HBx and HCC,the transient transfection cell models were established.The recombinant HBx and CYP3A4 plasmids were transfected into HepG2,HepG2.2.15 and Huh-7 cell lines to explore whether HBx could activate RAS-Raf-MEK-ERK signaling pathway,and the high expression of CYP3A4 could inhibit the activation by HBx.At the same time,the normal liver cell line L02 transient transfected cell models were utilized to simulate in vitro HBx-induced transformation process.In the process of transformation of normal cells to tumor cells,the induction of HBx on RAS-Raf-MEK-ERK signaling pathway,and the inhibition by CYP3A4 induced were dynamic monitored,then preliminary explored the intervention effects and mechanisms in the dynamic proces of CYP3A4 on HBx inducing normal liver cell carcinogenesis,as well as the degree increase of malignant tumor cells.ResultsSelect the clinical diagnosis and with a clear background in HCC patients with HBV infection in more than five years.The tissue samples were extracted and genetic markeed,then the drug-metabolizing enzymes which express rich or have a special function and the signal transduction pathways which related to the liver disease were detected.The results shown that,the drug-metabolizing enzyme CYP3A4 was reduced 27.09 times in HCC tumor tissue,compared with the adjacent normal tissues.Through the analysis of many tumor-related pathways of association and correlation tests,screening HCC correlated most closely with the signal transduction pathways RAS-Raf-MEK-ERK signaling pathway.The output of western blotting experiments shows that the expression levels of HBx and CYP3A4 in hepatoma cells transfected with HBx or CYP3A4plasmid were significantly increased.That indicats the recombinant plasmid HBx and CYP3A4 plasmid was successfully constructed,the plasmids could stably express in cells.L02 while the normal liver cell lines transfected with HBx concentration-dependent.After transfection of the recombinant plasmid,the expression levels of HBx and CYP3A4 were significantly increased.Especially in transfected L02 gradient model,HBx protein expression levels will be increased with the increasing concentration of HBx in cells.And the protein expression level of CYP3A4 gradually decreased.The test results for the RAS-Raf-MEK-ERK signaling pathway showed,HBx can effectively activate the RAS-Raf-MEK-ERK signaling pathway.The activated RAS were detected when HBx high expression.Meanwhile the major element of RAS-Raf-MEK-ERK signaling pathway,such as the protein expression levels of Raf-1,phospho-Raf-1,MEK 1/2,ERK 1/2,PP2A and KSR are also elevated.However,with the subsequent rise protein expression level of CYP3A4,the activated RAS protein and the protein expression levels of Raf-1,phospho-Raf-1,MEK 1/2,ERK 1/2,PP2A and KSR were significantly decreased.Similarly,in the normal liver cells L02 transient transfection model,HBx protein with a gradient of increased protein expression in the cells activated the expression of RAS,Raf-1,phospho-Raf-1,MEK 1/2,ERK 1/2,PP2A and KSR.But the high expression of CYP3A4,could effectively suppressed activation of HBx.RAS-Raf-MEK-ERK signaling pathway involved in a variety of cell physiological and biochemical functions,such as growth,proliferation,differentiation,apoptosis,migration and so on.It has been reported,RAS proteins regulate cell growth,proliferation,differentiation important signal element in the development of tumors,RAS is the abnormal proliferation and migration of tumor enhancement key incentive.The extracellular signal-regulated as a downstream effector kinase ERK(ERK)in cancer cells also had an outstanding physical contributions.Epithelial cells-interstitial conversion(EMT),is the epithelial cells through a specific program into biological processes having mesenchymal phenotype cells,the biological response is subsequent RAS-Raf-MEK-ERK signaling pathway activated.By wound-Healing Assay and transwell experiment,HBx could impact on the migration and invasion of liver cancer cell.The results showed that,when inrise the expressiong of HBx in liver cancer cells,the three types of EMT markers,including E-cadherin,N-cadherin,Vimentin and Snail were all increased.The results of cell scratches and migration experiments indicated that the tumor cells acquired a stronger migration and invasion after HBx infection.But the results in HBx-CYP3A4 group,after transfected with CYP3A4 plasmid,the expression levels of the three types of EMT markers,E-cadherin,N-cadherin,Vimentin and Snail,was significantly lower,migration and invasion of tumor cells can also significantly decreased.That shows CYP3A4 can effectively reverse the HBx-induced EMT processConclusionsIn HBV-induced HCC,HBx,as a functional protein of HBV,could be effectively activated RAS protein in cells,and increased the protein expression of Raf-1,phospho-Raf-1,MEK 1/2,ERK 1/2,PP2A and KSR,thereby activating the complete RAS-Raf-MEK-ERK signaling pathway.And after RAS-Raf-MEK-ERK signaling pathway activated,especially the subsequent physiological effects caused the tumor cells epithelial-mesenchymal transition,increased the migration and invasion ability,improved the malignant degree of tumor cells.But with the CYP3A4 transfected,the activation RAS protein was inhibited,and the protein expression of Raf-1,phospho-Raf-1,MEK 1/2,ERK 1/2,PP2A,and KSR reduced correspondingly,the invasion and migration ability of tumor cells also declined.Therefore,CYP3A4,as the major drug metabolizing enzymes in liver,which high expression could effectively reversed HBx activation of RAS-Raf-MEK-ERK signaling pathway,and could reduce the migration and invasive ability induced by HBx.which suggested that as the main function of HBV,HBx could activate the RAS-Raf-MEK-ERK signaling pathway,effectively induce conversion of normal cells into tumor cells,simultaneously CYP3A4 could effectively suppressed the HCC occurrence and development process induced by HBx.The results in the present study would provide a new target and theoretical basis for the selection of therapeutic drugs of the HCC patients.