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Ding et al. Hepatoma Res 2018;4:12  I  http://dx.doi.org/10.20517/2394-5079.2018.07                                                   Page 3 of 8


               Enzyme-linked immunosorbent assay analysis
               Enzyme-linked immunosorbent assay (ELISA) detection of targets of interest was performed according to
               the manufacturers’ instructions. β2GPI was measured in groups A, B, D, and E; HBsAg in groups B, D and
               F; and tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and alpha fetal protein (AFP) in all groups.
                                                                                         [6]
               Once β2GPI reached the highest expression level, as determined from previous studies , cell supernatants
               from each group were collected for ELISA analysis. Triplicates of standards, samples and blank groups were
               prepared. The optical density (OD) value of each well was measured at 450 nm. Data were presented as
               means ± SD.


               Non-radioactive NF-κB EMSA and NF-κB relative quantification
               Assays were performed only with nuclear extracts according to the manufacturer’s instructions. Nuclear
               extracts (5 μg) were used for each reaction with 400 fmol bio-labeled (hot) oligonucleotide NF-κB probe
               (5’-AGT TGA GGG GAC TTT CCC AGGC-3’) and unlabeled (cold)-NF-κB probe (5’-AGT TGA GGG
               GAC TTT CCC AGGC-3’). Poly(dI-dC): poly(dI-dC) was used as a nonspecific competitor. A 25-fold
               molar excess of unlabeled homologous oligonucleotide was used as a specific competitor. Non-homologous
               oligonucleotide sequences were also used to validate the specificity of the binding of each transcription factor
               in the competition assays. Binding reaction resolved by 6.5% acrylamide/bis (30:1 ratio) electrophoresis
               in 0.25× TBE on ice. The gel was transferred to nitrocellulose membranes in 0.5× TBE. The membrane
               was then UV crosslinked for 10 min, blocked with 1× blocking buffer for 30 min, and then incubated with
               streptavidin-HRP in blocking buffer (1:750) at room temperature for 30 min. The membrane was washed
               four times with 1× washing solution and was equilibrated with 1× equilibration solution for 5 min with
               shaking. Finally, the membranes were incubated with chemiluminescence substrate buffer, and the bands
               were visualized using Viagene CoolImager (Viagene Biotech Co., China). NF-κB relative quantification was
               based on relative activity of the combination of NF-κB and DNA. The last result was represented by ΔФ (gray
               value). The gray values of the image were measured after film exposure by the imaging system CoolImger.
               Data were presented as means ± SD.


               Statistical analysis
               SPSS 22.0 software was used for data processing and statistical analysis. Cell assay data were presented as
               means ± SDs and the variance was analyzed. Comparison between groups was measured using Fisher’s least
               significant difference (LSD) test. Differences were significant at P < 0.05.


               RESULTS
               Expression of β2GPI and HBsAg in transfected cells

               We used ELISA to measure expression of β2GPI and HBsAg 24h after transfection of recombinant plasmids
               in cell supernatants. β2GPI protein expression was found in group B, D, and E, significantly different from
               non-transfected, non-treated group A (P < 0.001). There were no differences in expression levels of β2GPI
               in groups B, D, and E (P > 0.05) suggesting similar transfection efficiency. HBsAg protein expression was
               found in groups B, D, and F. Expression was determined using a cutoff value (COV) that equal to the average
               absorbance value of the negative control (0.532). The absorbance of specimen ≥ COV indicated positive
               expression of HBsAg.


               Activation of NF-κB in β2GPI- and/rHBsAg-transfected cells following LPS stimulation
               A representative image of non-radioactive NF-κB EMSA in the six groups is shown in Figure 1, and NF-κB
               relative quantification was represented by gray value is shown in Figure 2. Groups B, C, D, E, and F induced
               differential levels of activation of NF-κB, with the highest relative activity of NF-κB observed in group D
               (1404.5 ± 11.28); this was significantly different compared with the other five groups (P < 0.05). The relative
               activity of NF-κB in group B was 914.57 ± 12.51, significantly higher than levels in groups A, C, E, and F
               (P < 0.05). The levels in group E (867.76 ± 6.27) and F (882.52 ± 7.92) were much higher than those of group
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