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Page 2 of 8                                                    Ding et al. Hepatoma Res 2018;4:12  I  http://dx.doi.org/10.20517/2394-5079.2018.07


               Keywords: Beta2-glycoproten I, hepatitis B surface antigen, lipopolysaccharide, liver cancer SMMC-7721, nuclear
               factor kappa B




               INTRODUCTION
               Beta2-glycoprotein I (β2GPI) also known as apolipoprotein H (apoH), is an abundant glycoprotein in the
                     [1]
               plasma . To date, most studies of β2GPI have focused on its role in anti-phospholipid antibody-thrombosis
                        [2,3]
                                                                                           [4]
                                                                                                        [5]
               syndrome , lipid metabolism, coagulation, and/or regulation of the fibrinolysis system . Mehdi et al.
               found hepatitis B surface antigen (HBsAg) bound β2GPI, an interaction that has been of interest to our
               research group. We previously found that there was a substantially increased level of β2GPI in hepatitis
                                                          [6]
               B-related hepatocellular carcinoma (HCC) tissue . The combination of β2GPI and HBsAg substantially
                                                    [6]
               activated nuclear factor kappa B (NF-κB) , suggesting that β2GPI played a role in the pathogenesis of
               hepatitis B-related HCC.
                            [7]
               A recent study  showed that lipopolysaccharide (LPS) specifically interacted with β2GPI, activating NF-κB
               via toll-like receptor 4 (TLR4) signaling pathway in macrophages. NF-κB is a pleiotropic transcription factor
                                                                                   [8]
               involved in inflammation-associated tumor promotion and progression in HCC . Most hepatitis B-related
               liver cancer patients experience dysbacteriosis, resulting in increased levels of and sensitivity to LPS. In the
               present study, we further examined whether LPS enhanced the effect of β2GPI and HBsAg on activation of
               NF-κB, as well as the expression of cytokine factors in the liver cancer cells.


               METHODS
               Experimental groups
               The human hepatoma cell line SMMC-7721 maintained in our laboratory were gifts from the central
               laboratory of the First Affiliated Hospital of Jilin University. The cells were incubated with Iscove’s modified
               Dulbecco’s medium (IMDM) culture media purchased from Gibco, containing 10% fetal bovine serum (FBS),
               and maintained at 37 °C in a 5% CO  incubator. All cells were grown to adherence and were passaged every
                                              2
               2-3 days. Cells in the logarithmic growth phase were selected for experimental use. SMMC-7721 cells were
               divided into six experimental groups. Group A was the control group, neither transfected nor treated; group
               B was co-transfected with β2GPI- and HBsAg plasmids without LPS treatment; group C was treated with
                                                          [9]
               500 μL (100 ng/mL) LPS and incubated for 6 h ; group D was transiently cotransfected with β2GPI-
               and HBsAg plasmids after treatment with 500 μL (100 ng/mL) LPS and incubated for 6 h; group E was
               transiently β2GPI-transfected after treatment with 500 μL (100 ng/mL) LPS and incubated for 6 h; group F
               was transiently HBsAg-transfected after treatment with 500 μL (100 ng/mL) LPS and incubated for 6 h.

               Cell transfection
               Groups B, D, E, and F were respectively transfected. The vector pcDNA3.1(-) was obtained from Invitrogen.
               The pcDNA3.1(-)-beta2-GPI and pcDNA3.1(-)-HBsAg eukaryotic expression plasmids were constructed
               previously in our laboratory. The recombinant plasmids, pcDNA3.1(-)-β2GPI, or pcDNA3.1(-)HBsAg at
               1 μg/well, and both at 3 μg/well (1:3) were dissolved in 50 μL IMMD basal media that was mixed to become
               Solution A. 2 μL FuGENE HD transfection reagent was dissolved in 50 μL IMMD basal media, mixed gently,
               incubated at room temperature for 5 min, labeled as Solution B. Solution A and Solution B were mixed
               gently to become Solution C, incubated at room temperature for 20 min. The cells were washed 3 times in
               serum-free IMMD culture media, and Solution C was slowly added to the cells that were incubated at 37 °C
               in a 5% CO  incubator. Transfection media was removed after 6-8 h and was replaced with 500 μL 10% FBS
                         2
                                                                                             [6]
               IMDM media. Cell supernatants were collected at 24 h after transfection. A previous study  from our lab
               found β2GPI protein expression was the highest 24 h after transfection.
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