Application Of ITRAQ In Hepatic Diseases

Hepatitis B virus infection is the most serious health problem around thewhole world, and is the main pathogenic factor causing chronic hepatic diseaseincluding chronic hepatitis B (CHB), liver fibrosis and hepatocellularcarcinoma(HCC).But there aren't effective therapy with CHB and HCC.Theantiviral drugs can not stop HBV replication completely, and the effect is notdesirable because of the side effect and drug resistence. HCC can not be resectedas they were always found at progressiv stage. Multiple drug resistence limits thechemotherapy effect. So if we can eliminate the HBV from host cells and reducethe incidence of resistence to chemotherapy in HCC, it is hopeful to stop theprogression to liver fibrosis or even HCC in CHB patient and reduce the mortalitysubstantially. With the development of proteomics technology such asmass-spectrometric and stable isotope label techniques, protein function can beanalysed in a brand new platform. We aim to solve the problems arising fromclinical medicine with iTRAQ coupled with MS technique using HepG2.2.15cellmodel and5-FU resistant BEL7402/5-FU cell line. We hope to identify proteomicsalteration raised by HBV replication and HCC drug resistence and find the keyproteins in it which can provide new clues in drug target search.In the HepG2and HepG2.2.15group,170differentially expressed proteinswere identified. Several differentially expressed proteins were further validated byWestern blot and real-time quantitative RT-PCR analysis. We demonstrated that thesuppression of HSP27expression in HepG2.2.15caused a nearly2-fold increase ofHBV production compared to the controls. In addition, the levels of both HBsAgand HBeAg in culture medium were also significantly increased. Furthermore,over-expression of HSP27in HepG2.2.15led to a marked decrease in HBV production, suggesting that HSP27functions as an endogenous anti-HBV factor.Increased mRNA levels of type â… interferon and five members in interferonstimulated genes were observed when HSP27were transfected into HepG2.2.15.Besides, expression of HSP27in70cases of HBV(-) paracancer liver normal tissuefrom HCC patient and90cases of HBV(+) paracancer tissue from HCC patientwere verified with immunohistochemistry.In the BEL7402and BEL7402/5-FU group,52differentially expressedproteins were identified. BEL7402. Parts of differentially expressed proteins werefurther validated by Western blot and real-time quantitative RT-PCR analysis,which has the same tendency with the MS results. The sensitivity ofBEL7402/5-FU to5-Fu, cisplatin, and adriamycin was increased when ANXA3was knocked down with siRNA transfection. Besides, expression of ANXA3in60paired liver tissue from HCC patient and paracancer tissue normal control wereverified with immunohistochemistry.Our study represents the first successful application of iTRAQ technology forHBV replication and MDR mechanisms analysis in HCC. Many of thedifferentially expressed proteins identified had not been linked to these twoprocesses before, which provide valuable information for further understanding ofthe mechnism.