Quantitative Proteomics Study On The Malignant Transformation Of Liver Cell Induced By Hepatitis B Virus X Gene

Primary liver cancer is the fifth most common cancer worldwide and the third mostcommon cause of cancer mortality. There are about700,000new cases worldwide every year.At present HCC is largely a disease of the third world, especially Southeast Asia andsub-Saharan Africa, where its prevalence exceeds that of occidental countries by many fold.China alone accounts for more than50%of the world’s HCC cases (age-standardizedincidence rate: male,35.2/100,000; female,13.3/100,000). HBV is implicated in the aetiologyof as much as54%of the HCC that occurs with such high frequency in Chinese populations.Although the obligative HBV vaccine immunization makes China HBV infected amount from9.75%to7.18%, the treatment and prevention of HBV patients is still a public health securityproblem urgently to be solved in China because of the larger population base and laggingeffect of existing infections occuring HCC.The HBV genome consists of four overlapping open reading frames encoding the four viralproteins, HBsAg, HBcAg, HBp and HBx. Of HBx function is complicated, as a kind ofmultifunctional virus regulatory factor, and promotes the liver cells malignant transformation.HBx was confirmed to participate the transcriptional regulation of virus and host cell, signaltransduction, protein degradation and other biological processes using yeast two-hybridsystem (Y2H), chromatin immune co-precipitation (CHIP), expression profile microarray andCo-Immunoprecipitation (Co-IP) method, which further confirmed the complexity anddiversity of HBx fuction. Although in the greater achievements in HBx research, there weremany deficiencies in the research strategy, experimental model and model of disease states.The strategy was mainly confined to the local signal pathway, not systematic, integratedanalysis, or the systematic research had only focused on gene mRNA level and ignored thechange of protein abundance. The patients’ tissue samples or in vitro cell line were as theexperimental models, which usually ignored the interference of microenvironment and geneticheterogeneity on HBx pathogenic mechanism research. Besides, the underlying mechanism ofHBx-mediated the process from liver inflammation to HCC remained obscure.Hence, the present study would strive to elucidate the mechanism of HBx-inducedmalignant transform process from inflammation to HCC stages using the high coverage andaccuracy of quantitative proteomics method. We performed the proteomics analysis of livertissues from12-month (12M) old HBx transgenic mice, which displayed mild inflammation;24-month (24M) old HBx transgenic mice, which exhibited HCC; and respectivenontransgenic littermates were as control, which would avoid the disturbance of geneticheterogeneity and individual life history on this research. Using F2generation SILAC mouseas internal standard, it would ensure the high precision quantitative analysis. At the same time, we screen the HBx interaction proteins (HIPs) using CO-IP combining proteomics methods.Combining HIPs with quantitative proteomics results, we will reconstruct the"HBx-HIPs-signal effect" signal network diagram.The present study firstly developed the overall SILAC labeling method for mouse model inChina. The C57BL/6mouse was raised with diet containing the [13C6]-Lysine and arrived atthe full labeling efficiency for proteome in the metabolism process. The incorporate labelingefficiency of F2SILAC mouse was beyond95%by the quantitative analysis for differentorgans and tissues using LC-MS/MS. The labeling efficiency of liver tissue arrived at96.34%±0.90%and met with the requirement of HBx transgenic mice study, which providedthe necessary condition for the high precision quantitative proteomics study.A total of162proteins were identified In the HIPs study. The GO analysis showed thatproteins were related to the cytoskeleton, mitochondrial function, lipid metabolism,transcriptional regulation, cell cycle and RNA splicing.47had also been identified as HIPs inthe previous study, and those were account for30%of162identified proteins, which showedthe excellent feasibility on the protein-protein interactions (PPIs) study using proteomicstechnology. The other115proteins were firstly identified in this study, demonstrating the hugeadvantage of PPIs study by proteomics method. In the identified162HIPs,37of the proteinsbelong to the largest functional group of cytoskeleton-related proteins, including MYL6B,ACTR3B, β-catenin, KIF11, KIF5C, AMOT, and SVIL, which promoted proliferation,migration, formation of tubular structure and the number of pseudopodia to increase tumorangiogenesis. The second largest protein group in HBx interactome contained32proteins,including GGCT, DUT, APRT and CAMK2D, which mediated the amino acids, nucleic acids,and RNA splicing process. Interestingly, not only HBx binded the rate-limiting enzyme ofβ-fatty acid oxidation process, ACSF2and HADH, but also binded three kinds of lipidcatabolic enzymes, SCP2, PNLIP and LCN1, affecting the liver lipid metabolism process.This was main cause of HBV-mediated liver steatosis and inflammation process.The quantitative proteomics results showed that a total of4,744proteins were identified inall group (FDR<1%). The identified proteins number of12M,24M HBx and their littematessample were3491,3464and3442,3298, respectively. A total of2,504overlapping proteinswere identified across all groups. In the quantitative research, the quantified overlap between12M HBx and12M WT sample was2816proteins, the SD value of all data distribution was0.20. The quantified overlap between24M HBx and24M WT sample was2731proteins, theSD value of all data distribution was0.42. The SD value of24M HBx sample was increasedcompared to that of12M HBx sample (P<0.05). It indicated that liver heterogeneity hadgradually increased in the HBx-mediated process from inflammation to HCC. By setting2-fold change as the threshold for significant up-or downregulation expression of proteins inthis study, we identified15upregulated and7downregulated proteins in12M HBx samplescompared with their littermates. We also identified72upregulated and25downregulatedproteins in24M HBx samples compared with their littermates. The biological process analysisshowed that muscle system process was heavily affected in12M samples. It might be related to hepatic stellate cell activation-triggered extracellular matrix fibrosis.The biological processanalysis showed that oxidation reduction, muscle tissue development, transcriptional activity,cytoskeleton, carbohydrate metabolism, lipid metabolism, small G protein signaling pathwayswere significantly enriched in24M HBx samples. Especially, we respectively noticed the3and15up-regulation expression proteins related to the CDC42signaling pathway. Itsuggested that CDC42signaling activation migt be a main cause for HBx-mediated processfrom inflammation to HCC. Although there was not a clue to mitochondrial dysfunction,4related lipid metabolism proteins were found, such as ADFP, DHTKD1, AKR1C18and CES2.At the HBx-mediated the process from inflammation to HCC,18related mitochondrialfunction proteins were found such as NDUFA5, LDHD, IVD and DHTKD1. Mitochondrialwas main organelle of lipid metabolism, these results suggested that HBx-induced lipidmetabolism disorder and mitochondrial dysfunction might also be a main cause of liverinflammation progress to HCC.By integrating the HIPs with the quantitative proteomics results, we found that the two datasets had favourable consistency in HBx-mediated cytoskeleton and mitochondrial function.Therefore, we put forward the scientific assumption that HBx induced inflammation and HCCin mice liver through dysregulation of cytoskeletal remodeling and lipid metabolism. Byimmunohistochemistry and Western blot validation experiments, we further confirmed theaccuracy of the quantitative proteomics data.Based on the similar difference proteins of HBx-induced inflammation and HCC stage, wescreened the cytoskeleton related protein CFL1and the lipid metabolism related proteinADFP as the candidate biomarkers for occurrence and development of HBx-mediated liverdisease and had conducted the thorough research. We analyzed the CFL1and ADFPexpression level of clinical HBV-HCC and non HBV-HCC tissue samples byimmunohistochemistry, and found that expression level of two proteins in HBV-HCC tissuesamples were significantly increased compared with those of non HBV-HCC tissue samples.These results had confirmed that CFL1and ADFP had the conservative property andspecificity as potential biomarker for HCC. According to the early prognosis and diagnosis ofHCC biomarkers with necessary elements for high specificity, sensitivity, reproducibility andpatient compliance, we futhur analyzed the protein abundance of CFL1and ADFP in serumsamples of HBV, HBV-HCC and HCV patients, and then we found that ADFP abundance wassignificantly increased in serum samples of HBV and HBV-HCC, however, the CFL1was notidentified in serum samples. Hence, ADFP could be a potential biomarker for early prognosisand diagnosis of HBV-HCC.To sum up, based on our high coverage and precision quantitative proteomics technologyplatform, the present study systematically analyzed the HBx fuction using HBx transgenicmouse and SILAC mouse as research model and quantitative internal reference. We hadelucidated the molecular mechanism that HBx-mediated the dysregulation of cytoskeletonremodeling and lipid metabolism might be pivotal factors in HBx-mediated progression frominflammation to HCC. At the same time, we also futhur confirmed that ADFP might be a potential biomarker for early prognosis and diagnosis of HBV-HCC by a large number ofclinical samples analysis. Our study not only enriched the biological function of HBx andmolecular mechanism of HBx-induced the occurrence and development of liver disease, andalso provided the idea for diagnosis, treatment and prevention of HBV and therapeutic targets.