Preliminary Study On The Role Of ZIP8 In HepG2 Cells To HCV Core Immune Response Process

Backgroud:Hepatitis C virus(HCV)infection is considered to be the leading cause of cirrhosis,chronic hepatitis and liver cancer worldwide.Patients with chronic hepatitis C first develop chronic inflammation leading in some to liver cirrhosis(20-40%)and subsequently(4-6%of patients)to hepatocellular carcinoma(10-40 years after inf'ections).Although it still remains controversial in the pathogenesis of hepatocellular carcinoma associated with HCV as to whether the virus plays a direct or an indirect role,there are strong evidences that the HCV proteins strengthen oncogenic transformation.The HCV-RNA gene encodes a polyprotein to produce virosomal structural proteins(core protein and glycoproteins E1 and E2)and nonstructural(NS)proteins(p7,NS2,NS3,NS4A.NS4B,NS5A and NS5B).In vivo.HCV core could mediate immune responses through various signaling pathways.leading to the occurrence of inflammation.Zinc transporters maintain the steady state of zinc in the cells,including those of the immune system through tight regulation of zinc influx,efflux,and distribution to intracellular organelles,which is a necessary condition for these metabolic and functional adjustments.ZIPs transporter increased the content of zinc in the cytoplasm,ZnTs transporter decreased the content of zinc in the cytoplasm,and the ZIP8 encoding the SLC39A8 gene belongs to ZIP transporters.Begum et al have found that expression of ZIP8 transporters is highly expressed in human immunostimulatory monocytes and differentiated macrophages.In transfected cells,ZIP8 is localized to lysosomes and can be induced by immune stimulation such as LPS.ZIP8 is closely related to the pathogenesis of osteoarthritis.In addition,ZIP8 can regulate the proinflammatory response through the negative feedback of NF-?B signaling pathway.Calcineurin(CaN)is a Ca2+/calmodulin(CaM)-dependent protein phosphatase that belongs to the family of protein phosphatase 2B and is widely distributed in many mammalian organs.Researchers have found that CaN could regulate the release of inflammatory cytokines through NF-?B signaling in vitro after LPS stimulation,such as IL-2,NO.In the CaN catalytic domain,some divalent metal ions such as Zn2+ and Fe2+ are essential elements.CaN catalytic domain can be stimulated by Mn2+ and Ni2+in vitro.Objective:Detect the ZIP8 mRNA expression levels in peripheral blood mononuclear cells(PBMCs)from the healthy normals,patients with CHC and cured patients with CHC to explore the relationship between CHC and ZIP8.Explore the role of ZIPS in HepG2 cells to HCV core immune response process by HepG2 cells treated with ZIP8 overexpression.ZIP8 interference and zinc deficiency,zinc plus.Methods:1.Clinical specimensWe detected the ZIP8 mRNA expression levels in PBMCs from the healthy controls(n=28).patients with CHC(n=39)and cured patients with CHC(n=49).2.The effect of HCV core on ZIP8 expression in HepG2 cellsHepG2 cells were cultured and transfected into pcDNA3.1 and pcDNA3.1-HCV core plasmids.The cells were harvested after 24h.The mRNA and protein expression levels of ZIP8 were detected by qRT-PCR and western blot.3.The role of ZIP8 in the HCV core immune response process(1)ZIP8?HCV core expression vectors were cotransfected into HepG2 cells,setting three groups:Vector?HCVc and HCVc + ZIP8.The cells were harvested after 24h.(2)ZIP8 RNA interference was transfected into HepG2 cells.After 48 h.HCV core expression vector was transfected into cells,setting three groups:Vector+NCsi?HCVc +NCsi and HCVc +ZIP8si.After incubation for 24 h,the cells were collected.4.The role of ZIP8 by regulating Zn2+ in the HCV core immune response process(1)HepG2 cells were pretreated with 2.5?M TPEN?50?M ZnCl2 for 1 h before HCV core transfection and incubation for another 24 h,setting four groups:Vector?HCVc?HCVc+ ZnCl2 and HCVc+TPEN.(2)ZIP8 RNA interference was transfected into HepG2 cells.After 48 h.HepG2 cells were pretreated with 50?M ZnC12 for 1 h before HCV core transfection and incubation for another 24 h,setting four groups:Vector+NCsi?HCVc +NCsi?HCVc +ZIP8si and HCVc +ZIP8si+ ZnCl2The above collected cells were used to extract RNA.intracellular total protein and intracellular nucleoprotein,and further to detect the mRNA expression levels of NF-KBp65?IL-6?IL-8 and TNF-a by qRT-PCR.Western blot was used to detect the protein expression levels of IKK??p-IKK? and the nuclear p-NF-KBp65.5.The effect of ZIP8 and Zn2+ on CaN activityThe total protein in each group was extracted.CaN assay kit was used to detect CaN activity in each group.6.Determination of intracellular zinc contentThe zinc ion content of the six experimental groups:HCVc?HCVc+ZIP8?HCVc+ZnCl2?HCVc+TPEN?HCVc+ZIP8si and HCVc+ZIP8si+ ZnCl2 were measured by zinc ion fluorescent probe.Results:1.Clinical specimensCompared to the healthy controls,the ZIP8 mRNA expression levels were significantly decreased in patients with CHC,and there was no significant difference in cured patients with CHC.Compared to patients with CHC,the ZIP8 mRNA expression levels were significantly increased in cured patients with CHC.2.HCV core up-regulated the ZIP8 expression in HepG2 cellsCompared with the control group,exogenous HCV core could significantly increase the expression of ZIP8 at mRNA and protein levels.3.The role of ZIP8 in the HCV core immune response processqRT-PCR results showed that ZIP8 overexpression could significantly decrease the mRNA expression levels of NF-KBp65?IL-6?IL-8 and TNF-?;ZIP8 RNA interference could significantly increase the mRNA expression levels of?NF-KBp65?IL-6?IL-8 and TNF-?.Western blot results showed that ZIP8 overexpression could significantly reduce the protein expression levels of IKK??p-IKK? and the nucleus p-NF-?cBp65:ZIP8 RNA interference could significantly increase the protein expression levels of IKK??p-IKK(3 and the nucleus p-NF-KBp65.4.The role of ZIP8 by regulating Zn2+ in the HCV core immune response processqRT-PCR results showed that 50?M ZnCl2 plus zinc treatment could significantly decrease the mRNA expression levels of NF-?Bp65?IL-6?IL-8 and TNF-a;2.5?M TPEN zinc deficiency could significantly increase the mRNA expression levels of NF-?Bp65?IL-6?IL-8 and TNF-a;After ZIP8 knocked out,50?M ZnCl2 plus zinc treatment could also significantly decrease the mRNA expression levels of NF-KBp65?IL-6.IL-8 and TNF-a.Western blot results showed that 50?M ZnCl2 plus zinc treatment could significantly reduce the protein expression levels of IKK??p-IKK?and the nucleus p-NF-KBp65;2.5?M TPEN zinc deficiency could significantly increase the protein expression levels of IKK??p-IKKp and the nucleus p-NF-KBp65;After ZIP8 knocked out,50pM ZnCl2 plus zinc treatment could also significantly decrease the protein expression levels of IKK??p-IKK? and the nucleus p-NF-KBp65.5.The effect of ZIP8 and Zn2+ on CaN activityZIP8 overexpression could significantly inhibit the increasing of CaN activity induced by HCV core;ZIP8 RNA interference could significantly induce the increasing of CaN activity;50?M ZnCl2 plus zinc treatment could significantly inhibite the increasing of CaN activity induced by HCV core;2.5?M TPEN zinc deficiency could significantly induce the increasing of CaN activity;After ZIP8 knocked out,50?M ZnCl2 plus zinc treatment could also significantly inhibit the increasingof CaN activity induced by HCV core.Thus.ZIP8 could inhibit the increasing of CaN activity induced by HCV core by regulating Zn2+6.Determination of intracellular zinc contentThe fluorescence intensity of HCVc+ZIP8 and HCVc+ZnCl3 corresponding to the control group HCVc was significantly enhanced;The fluorescence intensity of HCVc+ZIP8si and HCVc+TPEN corresponding to the control group HCVc was obviously weakened;The fluorescence intensity of HCVc +ZIP8si+ZnCl2 corresponding to the control group HCVc +ZIP8si was significantly enhanced.Conclusions:1.The results of clinical specimens showed that compared to the healthy controls,the ZIP8 mRNA expression levels were significantly decreased in patients with CHC,and there was no significant difference in cured patients with CHC.It was presumed that ZIP8 was associated with hepatitis C virus infection.2.In vitro experiments showed that ZIP8 overexpression and Zn2+ significantly inhibited the up-regulation of expression of inflammatory factors,the increased activity of NF-?B and CaN induced by HCV core,while silencing of ZIP8 and zinc deficiency showed the opposite results.It can be seen that ZIP8 can inhibit the occurrence of inflammation by regulating Zn2+,and the specific mechanism needs further study.