| Hepatocellular carcinoma (HCC) is the third most common cause of cancer in the world. It results in 598 000 deaths per year worldwide. Because of its poor prognosis, this number of deaths is almost the same as the number of cases being diagnosed each year (626 000). From a global perspective, the two most important risk factors for HCC are chronic hepatitis B and C infection. The geographic distribution patterns of HCC and hepatitis B virus (HBV) almost coincide with each other. The current situation in HCC therapy has urged clinical scientists to conduct extensive research on the genomics and signal pathway implicated in hepatocarcinogenesis, with the hope that a better understanding of HCC molecular pathogenesis will shed light on the new strategies of conquering this devastating disease.Beta2-glycoprotein I (beta2-GPI), also known as apolipoprotein H, a 50-kD protein and a glycoprotein of 326 aminuteso acids with a carbohydrate content of 17%, was apparently first described by Schultze et al. It is primarily synthesized in the liver and human plasma at ~200μg/mL. Beta2-GPI has been shown to be a cofactor for autoantibody recognition of anionic phospholipid antigen in anti-phospholipid syndrome (APS) and associated with thrombic events and recurrent miscarriage. The variability of beta2-GPI expression in individuals with various metabolic syndromes and disease states suggests that it may have clinical importance, and indeed, beta2-GPI was discovered to have many other functions. In 1994, Mehdi et al demonstrated that beta2-GPI could bind to recombinant hepatitis B surface antigen (rHBsAg), suggesting that it may facilitate entry of the virus into hepatocytes. The same group later chemically modified beta2-GPI to greatly enhance its reactivity with rHBsAg. Recently, it has been reported that the association of beta2-GPI with HBsAg is related to the presence of hepatitis B virus (HBV) markers, and that beta2-GPI binding activity for HBsAg was higher in sera from patients with virus in the active replication phase. In our past studies, we also verified that beta2-GPI specifically binds to rHBsAg. In addition, we provided the first proof that a protein exists on the SMMC-7721 HCC cell membrane which can specifically bind beta2-GPI. The binding protein was later identified as annexin II. Undoubtedly, there is an association between HBV infection and the development of HCC. Nevertheless, it is unclear whether either beta2-GPI and/or the beta2-GPI-HBsAg interaction play a role in the pathogenesis of HCC.NF-κB was originally identified as a nuclear factor specific to B cells bound to the B site of theκ-light chain gene enhancer, but it was later found also to be expressed in the liver epithelium, which can regulate hepatic cell proliferation and development. Classical NF-κB is a dimer composed of a p50 and a p65 subunit. In non-stimulated cells, NF-κB is sequestered in the cytoplasm through association with the inhibitory protein, IκB. The IκB proteins mask the nuclear localization signal of NF-κB, preventing its translocation into the nucleus. However, NF-κB can be activated and translocated from the cytoplasm into the nucleus, after exposure of cells to various stimuli, including viruses, antigens, stress factors and cytokines. Recent findings from several laboratories have implicated constitutive activation of the transcription factor NF-κB as one of the early key events involved in neoplastic progression of the liver.In the present study, we investigate the expression of beta2-GPI and its potential role in HBV-related HCC tissues and whether beta2-GPI and/or beta2-GPI and HBsAg interaction can activate the NF-κB pathway in vitroPurpose:This study investigates the effect and clinical significance of beta2-GPI combined HBsAg in hepatocellular carcinoma (HCC).Methods:1)Real time polymerase chain reaction (Real-time PCR) and double fluorescent immunostaining analysis were performed in 9 HCC parenchyma, 7 adjacent non-cancerous tissues and 5 control liver tissues from hepatitis B virus (HBV) infected patients using a beta2-GPI polyclonal antibody and an HBV surface antibody, to detect mRNA and protein expression of beta2-GPI gene in HCC tissues.2 ) Constructed the recombinant plasmid of pcDNA3.1(-)-beta2-GPI, pcDNA3.1 (-)-LHBsAg and pcDNA3.1(-)-SHBsAg using gene recombination technique. Stably- transfected cell lines SMMC-7721/beta2-GPI, which could maintain pcDNA3.1(-)-beta2-GPI plasmid in transfected cells, were screened and established.3)Hepatocellular carcinoma cells, SMMC-7721, were divided to nine groups. Group one: SMMC-7721/beta2-GPI cells were treated with 500μL HBsAg protein (300μg/mL) ; Group two: SMMC-7721 cells were treated with 500μL HBsAg protein (300μg/mL); Group three: SMMC-7721/beta2-GPI cells; Group four: normal control cells; Group five: SMMC-7721 cells transfected with control vector; Group six: SMMC-7721/beta2-GPI cells transfected with pcDNA3.1(-)-LHBsAg; Group seven: SMMC-7721/beta2-GPI cells transfected with pcDNA3.1(-)-SHBsAg; Group eight: SMMC-7721 cells transfected with pcDNA3.1(-)-LHBsAg; Group nine: SMMC-7721 cells transfected with pcDNA3.1(-)-SHBsAg.4)Beta2-GPI and alpha fetoprotein (AFP) proteins expression were also detected by ELISA kits in all groups.5)NF-κB activation was assessed by a non-radioactive electrophoretic mobility shift assay (EMSA) and immunofluorescence assay in SMMC-7721 HCC cells exposed to various treatments.6)IκB kinase inhibitor (BAY11-7082) and tyrosine kinase inhibitor (Piceatannol) were used to analyze the IκB activation for 0, 30, 60 and 90 minutes and for 0, 15, 30 and 60 minutes, respectively by non-radioactive EMSA.7)Phosphorylated amino acids and residues on the IκBαwere detected by co- immunoprecipitation assay followed by western blot analysis and immunofluorescence.Results:1)The plasmids pcDNA3.1(-)-beta2-GPI, pcDNA3.1(-)-LHBsAg and pcDNA3.1(-)- SHBsAg were constructed and confirmed by restriction enzyme digestion.2)The highest degree of co-localization of beta2-GPI and HBsAg proteins was seen in the endochylema and occurred at the nuclear border in the cancer tissues. Weak beta2-GPI protein staining was present in the endochylema, with a strong signal for HBsAg protein in HBV controls samples. Adjacent non-tumorous liver tissue samples also showed HBsAg staining but stronger beta2-GPI signals in the endochylema.3)In experiments with SMMC-7721 HCC cells, group three, eight and nine all had induced activation of NF-κB with the relative NF-κB DNA binding activities of 55.84, 42.77 and 40.75, respectively. However, the highest relative NF-κB DNA binding activity was observed in group seven (88.13). The second is group one(80.56).4)The mean AFP levels were significantly higher in group one, six and seven than other groups (P<0.05). But it appeared highest in group seven than others (P < 0.05), while no significant differences were seen between group four and five.5)BAY11-7082 inhibitor decreased NF-κB activation more obviously than Piceatannol inhibitor in the seven group with a relative NF-κB DNA binding activity of 88.13, 36.95, 24.09 and 17.18 on 0, 30, 60 and 90 minutes, respectively. The relative NF-κB DNA binding activity on Piceatannol inhibitor-treated group was 88.13, 63.58, 58.03, and 32.90 on 0, 15, 30 and 60 minutes, respectively.6)It was detected that only the serine-phosphorylated form of IκBαand phosphorylated residues were Ser32/36 in the cytoplasm.7)There was also detected that tyrosine form in thr group one.Conclusion:Beta2-GPI was strong expressed in the HBV-infected HCC. In vitro, beta2-GPI activates NF-κB in the HCC, but more efficient activation was observed by beta2-GPI and HBsAg combination. IκB kinase plays a significant role in the degradation of IκBα. Serine has phosphorylation effect and Ser32/36 residues are phosphorylated on the IκBα. It played a role in the development of HCC by activated NF-κB with beta2-GPI and HBsAg combination. These will be useful for pathogenesis and therapeutics of HBV infected hepatocellular carcinoma.
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