The Mechanism For Elevation Of Intracellular Calcium Level By Hepatitis B Virus X Protein By SOCs

Part ⅠConstruction and expression of a recombinant plasmid pEGFP-HBxObjectiveTo construct and express a recombinant plasmid pEGFP-HBx (p-HBx), and lay the foundation for further analyze the mechanism of the HBx protein-induced Hepatocellular carcinoma (HCC).Methods1Flag-HBX gene was amplified from pcDNA-Flag-HBx plasmid, and the HBX gene was detected by agarose electrophoresis.2The amplified HBX gene was modified by double digestion with restriction endonuclease. And the product was purified by agarose electrophoresis. The purified HBx gene fragment was inserted into an enhanced green fluorescent protein (PERES2-EGFP) expression vector to construct a recombinant plasmid pEGFP-HBx.3A HBx-expressing vector, pEGFP-HBx, was constructed and then transformed into E.coli, DH5ato clone and amplificate. The recombinant plasmid pEGFP-HBx (p-HBx) was validated by double digestion with restriction endonuclease and DNA sequencing analysis.4HepG2cells were transfected by pEGFP-HBx plasmid. The expression level of pEGFP-HBx was detected by fluorescence imaging and western blot.Results1The target gene band could be detected at about500bp by agarose electrophoresis. And the result was the same as the data of gene bank, which indicated that the cloned fragment was HBx gene.2The target gene bands could be detected at about500bp and5.3kb by agarose electrophoresis. The result showed the amplificate product had been inserted into PERES2-EGFP carrier.3The target protein band could be detected at about17KD by western blot. And the result was the same as the molecular weight of HBx protein. There were not detected the same phenomenon in control groups.4The green fluorescence could be detected in HepG2cells by fluorescence microscope, when these cells were transfected with pEGFP-HBx plasmid. There were not detected the same phenomenon in control groups. And the positive rate of transfected cells was about40%.ConclusionsHBx was cloned successfully into PIRES2-EGFP carrier. pEGFP-HBx plasmid was successfully structured and transfected into HepG2cells. And the expression level of pEGFP-HBx plasmid was high in HepG2cells. These results will accelerate the biological study of HBV-induced HCC. Part IIThe Hepatitis B virus X protein disturbs intracellular calcium signaling by binding with SOC componentsObjectiveThe present study aimed to determine whether HBx protein elevates the intracellular calcium through interacting with SOC components (STIM1and Orail) and to identify the targets of HBx protein for perturbing intracellular Ca2+homeostasis. To further reveal the molecular mechanism of HBx protein-induced HCC by elevation of intracellular calcium level.Methods1HEK293cells were cultured on collagen-coated tissue culture dishes using Dulbecco’s Modified Eagle Medium (DMEM) with10%fetal bovine serum (FBS) at37℃and5%CO2. For all experiments, media were replaced every3days. Cells were grown in60-mm dishes to about80%confluence and were transfected using Lipofectamine2000(Invitrogen) according to the manufacturer’s directions. All transfections were performed24hours after cell plating.2HEK293cells were observed by inverted microscope at24hours after transfection using pcDNA-HBx plasmids, and HBx protein was collected from HEK293cells and measured by western blot.3Co-IP was used to identify possible associations between HBx protein and SOCs components (STIM1and Orail). Precleared protein was obtained by incubating transfected HEK293cells. Briefly, Precleared protein was obtained by incubating transfected HEK293cells. Subsequently,50μl of1:1slurry of protein G-Sepharose beads were added. The samples were heated at95℃for5min before electrophoresis and blotted, and signal visualization was done using HA (rabbit polyclonal), Orail, or STIM1primary antibody respectively. As a negative control, protein samples were mock-immunoprecipitated with an irrelevant antibody and treated in the same way as described above. Proteins were visualized using an enhanced chemiluminescence (ECL) system (Bio-Rad).4HEK293cells were transfected using pEGFP-HBx and pcDNA-HA-Orail plasmids. Transfected HEK293cells were pretreated, and subsequently incubated with anti-Flag M2antibody and anti-HA antibody. Samples were washed in PBS and subsequently incubated with the secondary antibody. Images were captured by an Olympus Fluoview FV100laser scanning confocal microscope.5To determine the interactional site between HBx protein and Orial, Plasmid pGST-GEX-4T3served as backbone for the construction of GST-Orail or C-terminal (257-301aa), N-terminal (1-71aa) and loop domain (141-173aa) of Orail fusion proteins (GST at N terminus). Fusion proteins attached to glutathione-agarose beads were prepared according to protocols supplied in kit.Results1HEK293cells were observed by inverted microscope at24hours after transfection using pcDNA-HBx plasmids, HEK293cells grew well, and polygonous, semilucent and plump cells could be detected.2The target protein band could be detected at about17KD by western blot. And the result was the same as the molecular weight of HBx protein. There were not detected the same phenomenon in control groups.3The results of Co-IP assays showed that HBx protein could directly interacted with Orail to undergo dimerization, HBx protein did not show a physically interaction with the Orail.4HEK293cells were transfected with either pcDNA-GFP-HBx or pcDNA-HA-Orail plasmids, and the subcellular colocalization of these two proteins was detected by immunofluorescence assay. Most of GFP tagged HBx protein was expressed in cytoplasm, and less was located in the nucleus. The HA-Orail is labeled by the cy3-tagged secondary antibody that was located mainly in the cellular membrane. No detectable fluorescence was observed in untreated cells.5A recombinant GST-HBx-Orai1fusion protein was expressed in E. coli and immobilized on to the glutathione-agarose beads. Western blot revealed that Orai1-CT (257-301aa) could bind specifically to the GST-HBx protein, but not to the GST control. Furthermore, our results showed that it was the C-terminus of Orai1, neither the N-terminal nor loop domain of Orai1, did indeed interact with HBx protein in vitro. In similar experiments binding of STIM1to GST-HBx could not be detected.ConclusionsTransfected HEK293cells grew well. HBx gene could be expressed highly in transfected HEK293cells. HBx protein could directly interacted with the C-terminus of Orail to undergo dimerization, HBx protein did not show a physically interaction with the STIM1. And the colocalization of HBx and Orail was observed mostly in the cytoplasm and the cell membrane. The interaction between Orai1and HBx protein may induce the activation of SOCs, which may lead to the malignant transformation of liver cells. Part HIThe mechanism research of HBx protein elevating intracellular calcium by binding with Orail protienObjectiveThe present study aimed to determine whether HBx protein affects SOCE by interacting with Orail in HepG2cells, and clarify the possible mechanism of the HBx protein elevates intracellular calcium signaling. To identify the effect of HBx protein for perturbing intracellular Ca2+homeostasis on the hepatocyte proliferation, and reveal the role of HBx in the hepatocyte malignant transformation.Methods1HepG2cells were cultured on collagen-coated tissue culture dishes using Dulbecco’s Modified Eagle Medium (DMEM) with10%fetal bovine serum (FBS) at37℃and5%CO2. For all experiments, media were replaced every3days. Cells were grown in60-mm dishes to about80%confluence and were transfected using Lipofectamine2000according to the manufacturer’s directions. All transfections were performed24hours after cell plating.2For [Ca2+]i measurements, HepG2cells were culture in the cell culture medium with GdCl3, Inhibitor of Ca2+channels, and Fura-2-AM. Extracellular free Ca2+was eluted upon Ca2+-chelation with2mM EGTA and cells were washed several times with calcium-free Hepes-buffered salt solution (HBSS) at RT. Then HepG2cells expressing were pretreated with2μM thapsigargin in the absence of extracellular calcium to deplete stores,2mM calcium was then added to activate SOCE. The intracellular calcium levels were detected by a fluorescence microscope when GdCl3, the inhibitor of Ca2+channels, was used in experiment groups, and GdCl3, was not used in control groups.3For [Ca2+]i measurements, HepG2cells were culture in the cell culture medium with Fura-2-AM. Extracellular free Ca2+was eluted upon Ca2+-chelation with2mM EGTA and cells were washed several times with calcium-free Hepes-buffered salt solution (HBSS) at RT. Then HepG2cells expressing HBx were pretreated with2μM thapsigargin in the absence of extracellular calcium to deplete stores,2mM calcium was then added to activate SOCE. The intracellular calcium levels were detected by a fluorescence microscope when HBx gene was transfected into HepG2cells, and HepG2cells were not transfected in control groups.4For [Ca2+]i measurements, HepG2cells were culture in the cell culture medium with Fura-2-AM. Extracellular free Ca2+was eluted upon Ca2+-chelation with2mM EGTA and cells were washed several times with calcium-free Hepes-buffered salt solution (HBSS) at RT. Then HepG2cells expressing Orail and STIM1were pretreated with2μM thapsigargin in the absence of extracellular calcium to deplete stores,2mM calcium was then added to activate SOCE. The intracellular calcium levels were detected by a fluorescence microscope when pcDNA-HA-Orail and pcDNA-HA-STIM1plasmids were transfected into HepG2cells, and HepG2cells were not transfected in control groups.5For [Ca2+]i measurements, HepG2cells were culture in the cell culture medium with Fura-2-AM. Extracellular free Ca2+was eluted upon Ca2+-chelation with2mM EGTA and cells were washed several times with calcium-free Hepes-buffered salt solution (HBSS) at RT. Then HepG2cells expressing Orai1and HBx were pretreated with2μM thapsigargin in the absence of extracellular calcium to deplete stores,2mM calcium was then added to activate SOCE. The intracellular calcium levels were detected by a fluorescence microscope when pcDNA-HA-Orail and pEGFP-HBx plasmids were transfected into HepG2cells, and HepG2cells were transfected using pEGFP-HBx plasmids alone in control groups.6For [Ca2+]i measurements, HepG2cells were culture in the cell culture medium with Fura-2-AM. Extracellular free Ca was eluted upon Ca-chelation with2mM EGTA and cells were washed several times with calcium-free Hepes-buffered salt solution (HBSS) at RT. Then HepG2cells expressing Orail, HBx and STIM1were pretreated with2μM thapsigargin in the absence of extracellular calcium to deplete stores,2mM calcium was then added to activate SOCE. The intracellular calcium levels were detected by a fluorescence microscope when pcDNA-HA-Orail, pEGFP-HBx and pcDNA-HA-STIM1plasmids were transfected into HepG2cells, and HepG2cells were transfected using pcDNA-HA-Orailand pcDNA-HA-STIM1plasmids in control groups.7HepG2cells were observed by inverted microscope when pEGFP-HBx plasmids were transfected into HepG2cells, and the transfected cell viability was detected by CCk8reagent at Oh,12h,24h,48h after after transfection using pcDNA-HBx plasmids.Results1The inflow of extracellular calcium ions significantly increased when Gd3+was put into cell culture fluid by comparison with the control group (P<0.001).2The inflow of extracellular calcium ions significantly increased when HBx gene was transfected into HepG2cells by comparison with the control group (P<0.005).3The inflow of extracellular calcium ions significantly increased when STIM1gene and Orail gene were together transfected into HepG2cells by comparison with the control group (P<0.001).4The inflow of extracellular calcium ions significantly increased when HBx gene and Orail gene were together transfected into HepG2cells by comparison with the control group of transfection HBx alone(P<0.05).5HBx protein significantly increased cytosolic calcium signals in experimental groups by interaction with the component of SOCs(P<0.005).6The results of CCK8assays showed that HepG2cells proliferation significantly speeded up at24h,48h after transfection using pcDNA-HBx plasmids (P<0.05), while the number of transfected cell groups was less than that of control groups.ConclusionsHBx protein couples with the C-terminus of Orail and then may interact with STIM1to modulate SOCE, which may perturb intracellular calcium homeostasis. HBx protein may be a further contributor to the calcium-dependent activation of SOCs. HBx protein increases cytosolic calcium signals, which likely acts as an initiator for other HBx activities, such as regulation of cell proliferation.