HBV感染病人肝脏组织中IP-10的表达与HBx诱导其表达的分子机制研究
摘要
[目的]γ干扰素诱导蛋白10 (IP-10, CXCL10)是ELR的CXC趋化家族成员之一,也是CXCR3受体的一个配体,可以趋化NK细胞,活化的T细胞,DC细胞等。在HBV感染的病人外周血单个核细胞,肝窦内皮细胞,血小板中,IP-10的mRNA和蛋白质水平都显著增高。IP-10基因敲除的小鼠对稳定性嗜神经小鼠肝炎病毒的免疫反应减弱,伴随脑部CD4+和CD8+淋巴细胞浸润减少和炎性因子释放减少。因此,在HBV感染中,IP-10的诱导表达上调及其免疫效应是一非常重要的事件。但是,诱导IP-10表达上调的分子机制及其意义还不是十分清楚。在此研究中首先检测了不同病人肝组织中IP-10的表达,进一步通过细胞模型探索了HBx诱导趋化因子IP-10表达的分子机制及其白细胞趋化效应。
[方法]
1.在HBV感染病人肝脏组织中IP-10表达的检测
12例(8例男性,4例女性；年龄在32-65之间,平均年龄48±9)接受外科手术治疗的慢性HBV感染肝癌患者,7例接受外科手术治疗的非HBV感染肝癌患者和5例肝血管瘤患者肝脏正常组织分别从华中科技大学附属同济医院和协和医院收集,肝脏组织中IP-10mRNA和蛋白的表达分别采用real-time PCR和免疫组织化学方法检测。
2.转染不同质粒的HepG2细胞IP-10表达的检测
转染不同质粒的HepG2 (pBlue-HBV, pCMV-HBs, pCMV-HBc, pCMV-HBx,)细胞培养上清中的IP-10采用ELISA方法检测。
3.IP-10启动子荧光素酶报告基因活性检测
一系列5’端截短突变的IP-10启动子荧光素酶报告基因质粒和NF-κB突变的IP-10启动子荧光素酶报告基因质粒采用分子克隆技术构建,并与HBx真核表达质粒pCMV-HBx共转染HepG2细胞。48h后检测荧光素酶活性。
4.凝胶迁徙阻滞实验
转染不同质粒的细胞核抽提物与生物素标记的NF-κB1探针共同孵育,加入或不加入未标记的野生型NF-κB1探针或未标记的突变型NF-κB1探针,随后用6%聚丙烯酰胺凝胶在0.5×TBE中电泳,电泳后转膜,Image Station 4000R成像检测膜上化学发光信号。
5.染色质免疫共沉淀实验
HepG2细胞转染不同质粒后用1%甲醛交联,超声打断染色质,与anti-p65抗体,anti-p50抗体或同型对照IgG一起孵育共沉淀。从染色体-抗体复合物中分离出DNA用于PCR扩增,琼脂糖凝胶电泳分析。
6.激光共聚焦显微术
转染不同质粒的HepG2细胞爬片用冷甲醇固定,1%牛血清白蛋白封闭,anti-p65一抗孵育。FITC标记的二抗检测信号,激光共聚焦显微镜观察。
7.趋化实验
趋化实验采用5-μm孔径的24孔Transwell板进行。1×105外周血单个核细胞用200μl RPMI培养液悬浮加入到上室。1ml转染了相应质粒的HepG2细胞上清液加入下室。Transwell板在37℃,5%CO2培养箱中孵育4h后,对趋化的细胞HE染色,计数。
[结果]
1.HBV感染的肝脏组织中IP-10的表达
采用real-time PCR和免疫组织化学方法检测HBV感染肝脏组织中IP-10mRNA和蛋白表达结果提示,HBV阳性的肝癌组织中IP-10的mRNA和蛋白表达均显著高于HBV阴性肝癌组织以及肝血管瘤的正常肝脏组织,这些数据表明,在HBV感染的肝脏组织中IP-10的表达升高,可能在HBV诱导的肝脏炎症损伤中起着重要作用。
2. HepG2细胞中HBx蛋白诱导IP-10的表达
转染了HBV全长表达质粒(pBlue-HBV)后,HepG2细胞培养上清液中IP-10蛋白水平显著增高。为了检测HBV不同蛋白分子对诱导IP-10的作用,分别转染了编码不同HBV蛋白分子的真核表达质粒(pCMV-HBs, pCMV-HBc, pCMV-HBx),结果表明HBx蛋白可以显著增加IP-10的表达。IP-10启动子荧光素酶活性分析结果提示IP-10的启动子能被HBx蛋白呈剂量依赖性激活。
3.HBx诱导的IP-10表达上调是通过激活TRAF2/TAK1/NF-κB信号途径
为了探讨HBx诱导IP-10表达的机制,构建了一系列5’端缺失的IP-10启动子荧光素酶报告基因载体,并与HBx真核表达质粒pCMV-HBx共转染HepG2细胞。荧光素酶活性检测结果提示在IP-10启动子nt-190 to-96区域对HBx激活IP-10启动子起重要作用,进一步分析发现在这一区域有两个NF-κB结合位点(NF-κB1和NF-κB2)。NF-κB结合位点定点突变实验证实,IP-10启动子的NF-κB1结合位点对HBx诱导IP-10表达起作用。加入NF-κB的抑制剂SN50后,可抑制HBx诱导的IP-10表达。同时,NF-κB亚单位p65和p50的增加能够促进IP-10的表达。
为了探讨激活的NF-κB是否直接与IP-10启动子结合而实现对其调控,进一步进行了EMSA和CHIP实验。用生物素标记的IP-10启动子NF-κB1寡核苷酸探针检测结果显示,在转染了pCMV-HBx质粒的细胞核抽提物中NF-κB蛋白与DNA的结合增强；转染了pCMV-HBx质粒的细胞核抽提物,用anti-p50或anti-p65抗体进行免疫沉淀,沉淀物分离出的DNA能够扩增出177bp的IP-10启动子序列。以上结果显示,NF-κB能够与IP-10启动子上的NF-κB1结合位点直接结合。
为了进一步探讨HBx激活NF-κB的机制,western blot、启动子荧光素酶活性以及共聚焦显微镜术检测结果提示,在转染了HBx蛋白真核表达载体pCMV-HBx后,HepG2细胞中TAK1和IKKα的磷酸化水平增加。加入TAK1的抑制剂或转染TAK1的siRNA阻断TAK1的作用后,NF-κB和IP-10的启动子活性均被抑制。TRAF家族成员TRAF2阻断后也能阻碍NF-κB和IP-10的启动子活性。同时,在TAK1和TRAF2阻断后NF-κB亚单位p65的核转位被抑制。上述结果显示,HBx诱导的IP-10转录表达是通过激活TRAF2/TAK1/NF-κB信号通路而实现的。由此我们推测,HBx可能通过TLR信号传导,TAK1被激活,进而激活IKK,最终激活NF-κB。
4.HBx诱导IP-10表达上调增加外周血单个核细胞的趋化性
趋化实验结果表明,相比转染对照质粒,转染了HBx,NF-κB p50或NF-κBp65真核表达质粒的HepG2细胞上清液对外周血单个核细胞的趋化活性增加。加入抗IP-10的中和抗体后能减弱其对外周血单个核细胞的趋化。加入了NF-κB的抑制剂SN50后,能减弱转染HBx的细胞上清诱导外周血单个核细胞的趋化性。
[结论]本课题研究证明HBV阳性的肝癌组织中IP-10的mRNA和蛋白表达水平均显著高于HBV阴性肝癌组织以及肝血管瘤的正常肝脏组织,提示在HBV感染的肝脏组织中IP-10的表达可能在HBV诱导的肝脏炎症损伤中起着重要作用。在此基础上进一步通过转染了编码HBV不同蛋白基因的HepG2细胞模型,探讨了HBV诱导IP-10表达的机制,研究证明病毒蛋白HBx可诱导炎性趋化因子IP-10表达增加,其机制为病毒蛋白HBx通过激活TRAF2/TAK1/NF-κB信号通路,从而导致IP-10启动子的转录激活,IP-10的表达增加。由此推测,HBx诱导的IP-10等趋化因子的表达增加,趋化炎性细胞的浸润可能在导致肝脏免疫损伤中起重要作用。
[Objective] Interferon-y inducible protein 10 (IP-10, CXCL10) is a member of the non-ELR CXC chemokine family and a ligand for the CXCR3 receptor, chemoattracting for NK cells, activated T cells, and dendritic cells. The mRNA and protein levels of IP-10 in PBMCs, sinusoidal endothelium and plasma are all increased in patients infected hepatitis B. In IP-10-/- mice, immune responses to infection by neurotropic mouse hepatitis virus are reduced, concomitant with decreased CD4+and CD8+ lymphocyte trafficking to the brain and reduced production of inflammatory factors. Therefore, the induction of IP-10 is an important event for the development of hepatitis B. However, the molecular basis of how IP-10 is regulated for the induction and function exertion still remains unknown. In the present study, we explored the IP-10 expression in HBV-infected liver tissues and then defined underlying molecular mechanisms for HBx-induced IP-10 expression and leukocytes migration by cell line models.
[Methods]
1. IP-10 expression detection in HBV-infection patients
Twelve patients (8 male,4 female; age range 32-65 years, average 48±9) with chronic hepatitis B who underwent surgery treatment for cancers, seven patients with non-HBV infection underwent surgery for cancers and five normal control Hepatic hemangioma liver tissues were selected from the Union Hospital of Tongji Medical College for IP-10 detection in the liver tissues by real-time PCR and immunohistochemistry.
2. Plasimds transfection and IP-10 expression in HepG2 cells
HepG2 cells were transfected with relative plasmid (pBlue-HBV, pCMV-HBs, pCMV-HBc, pCMV-HBx,) and the supernatants IP-10 expressin was detected by ELISA.
3. IP-10 promoter luciferase reporter assays
A series of 5'deletions and NF-κB mutation of IP-10 luciferase promoter plasmids were constructed and co-transfected with pCMV-HBx, respectively, into HepG2 cells. Luciferase activity was measured 48 hrs after transfection.
4. Electrophoretic Mobility Shift Assay
Nuclear extracts transfected relative plasmids were incubated with the labeled NF-κB1 binding oligonucleotides in the presence or out the presence of either an unlabeled wild type NF-κB binding probe or a mutated probe and then were separated on 6% polyacrylamide gels with 0.5×TBE buffer and transferred to a nylon membrane. A chemiluminescent detection of membranes was scanned by Image Station 4000R.
5. Chromatin immunoprecipitation assay
HepG2 cells transfected with relative plasmids were cross-linked histones to DNA using 1% formaldehyde and sonicated to shear the chromatins. The sonicated chromatins were incubated with anti-p65 antibody, anti-p50 antibody or an isotype control IgG for 2 hrs. The DNA isolated from the chromatin-antibody complex was subject to PCR amplification and agarose gel electrophoresis.
6. Confocal Laser Scanning Microscopy
HepG2 cells transfected relative plasmids were cultured on coverslips. After fixed with ice-cold methanol, cells were blocked with 1% bovine serum albumin and probed with anti-p65 antibody. Signals were visualized with FITC-conjugated second antibody and micrographs were acquired with a BX51 system equipped with DP70.
7. Migration Assay
Migratory activity was quantified using 24-well Transwell inserts(5-μm pore size). PBLs (1×105) in 200μl of RPMI medium were added to the upper chamber.1 ml culture supernatant from the cells transfected with relative plasmids was added to the lower chamber. The chambers were incubated for 4 hrs at 37℃in 5% CO2, then the Transwell inserts were removed and the migration cells were HE stained and counted.
[Results]
1. IP-10 expression in HBV-infected liver tissues.
We analyzed the expression of IP-10 in HBV-infected liver tissues tissues, and found that the mRNA and protein levels of IP-10 were much higher in HBV positive liver cancer tissues, compared to HBV negative liver cancer tissues or normal Hepatic hemangioma liver tissues. These data suggested that IP-10 is elevated in HBV-infected liver tissue, and may play an important role in HBV-induced inflammation of the liver injury.
2. HBx induces IP-10 gene expression in HepG2 cells.
The protein levels of IP-10 in the supernatants in HepG2 cells were significantly increased after transfected with the full-length HBV gene (pBlue-HBV). Evaluated the effect of different HBV proteins we defined that the transfection of HBx-rather than HBs-or HBc-expressing vector increased IP-10 protein levels. By promoter luciferase activity assay, we found that IP-10 promoter was markedly activated by HBx protein in a dose-dependent manner.
3. HBx-induced IP-10 expression is mediated by TRAF2/TAK1/NF-κB signaling pathway
To investigate the mechanize of HBx-induced IP-10 expression, a series of 5' deletions of IP-10 promoter were constructed and co-transfected with pCMV-HBx, respectively, into HepG2 cells. Luciferase assay suggest that the sequence between nt -190 to-96 is critical for activation of IP-10 promoter by HBx protein. Coincidently, two NF-κB binding sites (NF-κB1, and NF-κB2) were found in this region. Afer mutation the two NF-κB binding sites, we confirmed that the NF-κB 1 binding site is required for the activation of IP-10 promoter regulated by HBx protein. The addition of NF-κB inhibitor SN50, blocks the effect of HBx on IP-10 induction. In parallel, the increase of NF-κB subunits p65 and p50 in HepG2 cells also augments IP-10 expression.
Next, we asked whether the activated NF-κB bound to IP-10 promoter directly. For this purpose, we performed EMSA and CHIP assay. Results showed the DNA binding activity of NF-κB was significantly increased in the cells transfected with pCMV-HBx detected by a biotin-labeled NF-κB1 binding oligonucleotides probe in the IP-10 promoter (-125 to-102) and a 177-bp DNA fragment in IP-10 promoter was amplified by PCR from DNA which isolated from cells transfected with pCMV-HBx and then immunoprecipitated with anti-p50 or anti-p65 antibody. These data suggested that the NF-κB directly binds to the NF-κB1 binding site in the IP-10 promoter.
To further defined the mechanism of HBx activated NF-κB activation. In this regard, by western blot, promoter luciferase activity and confocal assay we found after transfection of HBx plasmid into HepG2 cell line, the phosphorylation of TAK1 and IKKa was observed in HBx plasmid group. By addition TAK1 inhibitor or TAK1 siRNA, both NF-κB activity and IP-10 promoter activity were reduced. TRAF family member TRAF2 knockdown also impaired the NF-κB and IP-10 activity induced by HBx. Consistently, the inhibition of the nuclear translocation of NF-κB subunit p65 was defined after knockdown of TRAF2 or TAK1. Taken together, our data suggested that HBx-induced IP-10 expression might be mediated by TRAF2/TAK1/NF-κB signaling pathway.
4. HBx-induced IP-10 increases migration of peripheral blood leukocytes (PBLs)
We performed migration assay and found that PBLs exhibited decent increase of migratory activity in response to supernatants from cells transfected with plasmids expressing HBx, p50, or p65 comparing mock plasmids. The addition of neutralizing anti-IP-10 but not the control antibody to the supernatants significantly impaired this migration. Interestingly, NF-κB inhibitor SN50 treatment could decrease the chemotactic activity after transfection of pCMV-HBx. Together, these data suggested that HBx-induced IP-10 may mediate the migration of PBLs and NF-κB activity is involved in this process.
[Conclusions] Our study found that the mRNA and protein levels of IP-10 were much higher in HBV positive liver cancer tissues, compared to HBV negative liver cancer tissues or normal Hepatic hemangioma liver tissues, and may play an important role in HBV-induced inflammation of the liver injury. Then we transfected different HBV protein expressing plasmids into HepG2 cells and explored a novel molecular mechanism of HBV infection inducing IP-10 expression, which involves viral protein HBx affecting TRAF2/TAK1/NF-κB pathway, leading to the transactivation of IP-10 promoter. Our study provides insight into the migration of leucocytes in response to HBV infection, thus causing immune pathological injury of liver.
[方法]
1.在HBV感染病人肝脏组织中IP-10表达的检测
12例(8例男性,4例女性；年龄在32-65之间,平均年龄48±9)接受外科手术治疗的慢性HBV感染肝癌患者,7例接受外科手术治疗的非HBV感染肝癌患者和5例肝血管瘤患者肝脏正常组织分别从华中科技大学附属同济医院和协和医院收集,肝脏组织中IP-10mRNA和蛋白的表达分别采用real-time PCR和免疫组织化学方法检测。
2.转染不同质粒的HepG2细胞IP-10表达的检测
转染不同质粒的HepG2 (pBlue-HBV, pCMV-HBs, pCMV-HBc, pCMV-HBx,)细胞培养上清中的IP-10采用ELISA方法检测。
3.IP-10启动子荧光素酶报告基因活性检测
一系列5’端截短突变的IP-10启动子荧光素酶报告基因质粒和NF-κB突变的IP-10启动子荧光素酶报告基因质粒采用分子克隆技术构建,并与HBx真核表达质粒pCMV-HBx共转染HepG2细胞。48h后检测荧光素酶活性。
4.凝胶迁徙阻滞实验
转染不同质粒的细胞核抽提物与生物素标记的NF-κB1探针共同孵育,加入或不加入未标记的野生型NF-κB1探针或未标记的突变型NF-κB1探针,随后用6%聚丙烯酰胺凝胶在0.5×TBE中电泳,电泳后转膜,Image Station 4000R成像检测膜上化学发光信号。
5.染色质免疫共沉淀实验
HepG2细胞转染不同质粒后用1%甲醛交联,超声打断染色质,与anti-p65抗体,anti-p50抗体或同型对照IgG一起孵育共沉淀。从染色体-抗体复合物中分离出DNA用于PCR扩增,琼脂糖凝胶电泳分析。
6.激光共聚焦显微术
转染不同质粒的HepG2细胞爬片用冷甲醇固定,1%牛血清白蛋白封闭,anti-p65一抗孵育。FITC标记的二抗检测信号,激光共聚焦显微镜观察。
7.趋化实验
趋化实验采用5-μm孔径的24孔Transwell板进行。1×105外周血单个核细胞用200μl RPMI培养液悬浮加入到上室。1ml转染了相应质粒的HepG2细胞上清液加入下室。Transwell板在37℃,5%CO2培养箱中孵育4h后,对趋化的细胞HE染色,计数。
[结果]
1.HBV感染的肝脏组织中IP-10的表达
采用real-time PCR和免疫组织化学方法检测HBV感染肝脏组织中IP-10mRNA和蛋白表达结果提示,HBV阳性的肝癌组织中IP-10的mRNA和蛋白表达均显著高于HBV阴性肝癌组织以及肝血管瘤的正常肝脏组织,这些数据表明,在HBV感染的肝脏组织中IP-10的表达升高,可能在HBV诱导的肝脏炎症损伤中起着重要作用。
2. HepG2细胞中HBx蛋白诱导IP-10的表达
转染了HBV全长表达质粒(pBlue-HBV)后,HepG2细胞培养上清液中IP-10蛋白水平显著增高。为了检测HBV不同蛋白分子对诱导IP-10的作用,分别转染了编码不同HBV蛋白分子的真核表达质粒(pCMV-HBs, pCMV-HBc, pCMV-HBx),结果表明HBx蛋白可以显著增加IP-10的表达。IP-10启动子荧光素酶活性分析结果提示IP-10的启动子能被HBx蛋白呈剂量依赖性激活。
3.HBx诱导的IP-10表达上调是通过激活TRAF2/TAK1/NF-κB信号途径
为了探讨HBx诱导IP-10表达的机制,构建了一系列5’端缺失的IP-10启动子荧光素酶报告基因载体,并与HBx真核表达质粒pCMV-HBx共转染HepG2细胞。荧光素酶活性检测结果提示在IP-10启动子nt-190 to-96区域对HBx激活IP-10启动子起重要作用,进一步分析发现在这一区域有两个NF-κB结合位点(NF-κB1和NF-κB2)。NF-κB结合位点定点突变实验证实,IP-10启动子的NF-κB1结合位点对HBx诱导IP-10表达起作用。加入NF-κB的抑制剂SN50后,可抑制HBx诱导的IP-10表达。同时,NF-κB亚单位p65和p50的增加能够促进IP-10的表达。
为了探讨激活的NF-κB是否直接与IP-10启动子结合而实现对其调控,进一步进行了EMSA和CHIP实验。用生物素标记的IP-10启动子NF-κB1寡核苷酸探针检测结果显示,在转染了pCMV-HBx质粒的细胞核抽提物中NF-κB蛋白与DNA的结合增强；转染了pCMV-HBx质粒的细胞核抽提物,用anti-p50或anti-p65抗体进行免疫沉淀,沉淀物分离出的DNA能够扩增出177bp的IP-10启动子序列。以上结果显示,NF-κB能够与IP-10启动子上的NF-κB1结合位点直接结合。
为了进一步探讨HBx激活NF-κB的机制,western blot、启动子荧光素酶活性以及共聚焦显微镜术检测结果提示,在转染了HBx蛋白真核表达载体pCMV-HBx后,HepG2细胞中TAK1和IKKα的磷酸化水平增加。加入TAK1的抑制剂或转染TAK1的siRNA阻断TAK1的作用后,NF-κB和IP-10的启动子活性均被抑制。TRAF家族成员TRAF2阻断后也能阻碍NF-κB和IP-10的启动子活性。同时,在TAK1和TRAF2阻断后NF-κB亚单位p65的核转位被抑制。上述结果显示,HBx诱导的IP-10转录表达是通过激活TRAF2/TAK1/NF-κB信号通路而实现的。由此我们推测,HBx可能通过TLR信号传导,TAK1被激活,进而激活IKK,最终激活NF-κB。
4.HBx诱导IP-10表达上调增加外周血单个核细胞的趋化性
趋化实验结果表明,相比转染对照质粒,转染了HBx,NF-κB p50或NF-κBp65真核表达质粒的HepG2细胞上清液对外周血单个核细胞的趋化活性增加。加入抗IP-10的中和抗体后能减弱其对外周血单个核细胞的趋化。加入了NF-κB的抑制剂SN50后,能减弱转染HBx的细胞上清诱导外周血单个核细胞的趋化性。
[结论]本课题研究证明HBV阳性的肝癌组织中IP-10的mRNA和蛋白表达水平均显著高于HBV阴性肝癌组织以及肝血管瘤的正常肝脏组织,提示在HBV感染的肝脏组织中IP-10的表达可能在HBV诱导的肝脏炎症损伤中起着重要作用。在此基础上进一步通过转染了编码HBV不同蛋白基因的HepG2细胞模型,探讨了HBV诱导IP-10表达的机制,研究证明病毒蛋白HBx可诱导炎性趋化因子IP-10表达增加,其机制为病毒蛋白HBx通过激活TRAF2/TAK1/NF-κB信号通路,从而导致IP-10启动子的转录激活,IP-10的表达增加。由此推测,HBx诱导的IP-10等趋化因子的表达增加,趋化炎性细胞的浸润可能在导致肝脏免疫损伤中起重要作用。
[Objective] Interferon-y inducible protein 10 (IP-10, CXCL10) is a member of the non-ELR CXC chemokine family and a ligand for the CXCR3 receptor, chemoattracting for NK cells, activated T cells, and dendritic cells. The mRNA and protein levels of IP-10 in PBMCs, sinusoidal endothelium and plasma are all increased in patients infected hepatitis B. In IP-10-/- mice, immune responses to infection by neurotropic mouse hepatitis virus are reduced, concomitant with decreased CD4+and CD8+ lymphocyte trafficking to the brain and reduced production of inflammatory factors. Therefore, the induction of IP-10 is an important event for the development of hepatitis B. However, the molecular basis of how IP-10 is regulated for the induction and function exertion still remains unknown. In the present study, we explored the IP-10 expression in HBV-infected liver tissues and then defined underlying molecular mechanisms for HBx-induced IP-10 expression and leukocytes migration by cell line models.
[Methods]
1. IP-10 expression detection in HBV-infection patients
Twelve patients (8 male,4 female; age range 32-65 years, average 48±9) with chronic hepatitis B who underwent surgery treatment for cancers, seven patients with non-HBV infection underwent surgery for cancers and five normal control Hepatic hemangioma liver tissues were selected from the Union Hospital of Tongji Medical College for IP-10 detection in the liver tissues by real-time PCR and immunohistochemistry.
2. Plasimds transfection and IP-10 expression in HepG2 cells
HepG2 cells were transfected with relative plasmid (pBlue-HBV, pCMV-HBs, pCMV-HBc, pCMV-HBx,) and the supernatants IP-10 expressin was detected by ELISA.
3. IP-10 promoter luciferase reporter assays
A series of 5'deletions and NF-κB mutation of IP-10 luciferase promoter plasmids were constructed and co-transfected with pCMV-HBx, respectively, into HepG2 cells. Luciferase activity was measured 48 hrs after transfection.
4. Electrophoretic Mobility Shift Assay
Nuclear extracts transfected relative plasmids were incubated with the labeled NF-κB1 binding oligonucleotides in the presence or out the presence of either an unlabeled wild type NF-κB binding probe or a mutated probe and then were separated on 6% polyacrylamide gels with 0.5×TBE buffer and transferred to a nylon membrane. A chemiluminescent detection of membranes was scanned by Image Station 4000R.
5. Chromatin immunoprecipitation assay
HepG2 cells transfected with relative plasmids were cross-linked histones to DNA using 1% formaldehyde and sonicated to shear the chromatins. The sonicated chromatins were incubated with anti-p65 antibody, anti-p50 antibody or an isotype control IgG for 2 hrs. The DNA isolated from the chromatin-antibody complex was subject to PCR amplification and agarose gel electrophoresis.
6. Confocal Laser Scanning Microscopy
HepG2 cells transfected relative plasmids were cultured on coverslips. After fixed with ice-cold methanol, cells were blocked with 1% bovine serum albumin and probed with anti-p65 antibody. Signals were visualized with FITC-conjugated second antibody and micrographs were acquired with a BX51 system equipped with DP70.
7. Migration Assay
Migratory activity was quantified using 24-well Transwell inserts(5-μm pore size). PBLs (1×105) in 200μl of RPMI medium were added to the upper chamber.1 ml culture supernatant from the cells transfected with relative plasmids was added to the lower chamber. The chambers were incubated for 4 hrs at 37℃in 5% CO2, then the Transwell inserts were removed and the migration cells were HE stained and counted.
[Results]
1. IP-10 expression in HBV-infected liver tissues.
We analyzed the expression of IP-10 in HBV-infected liver tissues tissues, and found that the mRNA and protein levels of IP-10 were much higher in HBV positive liver cancer tissues, compared to HBV negative liver cancer tissues or normal Hepatic hemangioma liver tissues. These data suggested that IP-10 is elevated in HBV-infected liver tissue, and may play an important role in HBV-induced inflammation of the liver injury.
2. HBx induces IP-10 gene expression in HepG2 cells.
The protein levels of IP-10 in the supernatants in HepG2 cells were significantly increased after transfected with the full-length HBV gene (pBlue-HBV). Evaluated the effect of different HBV proteins we defined that the transfection of HBx-rather than HBs-or HBc-expressing vector increased IP-10 protein levels. By promoter luciferase activity assay, we found that IP-10 promoter was markedly activated by HBx protein in a dose-dependent manner.
3. HBx-induced IP-10 expression is mediated by TRAF2/TAK1/NF-κB signaling pathway
To investigate the mechanize of HBx-induced IP-10 expression, a series of 5' deletions of IP-10 promoter were constructed and co-transfected with pCMV-HBx, respectively, into HepG2 cells. Luciferase assay suggest that the sequence between nt -190 to-96 is critical for activation of IP-10 promoter by HBx protein. Coincidently, two NF-κB binding sites (NF-κB1, and NF-κB2) were found in this region. Afer mutation the two NF-κB binding sites, we confirmed that the NF-κB 1 binding site is required for the activation of IP-10 promoter regulated by HBx protein. The addition of NF-κB inhibitor SN50, blocks the effect of HBx on IP-10 induction. In parallel, the increase of NF-κB subunits p65 and p50 in HepG2 cells also augments IP-10 expression.
Next, we asked whether the activated NF-κB bound to IP-10 promoter directly. For this purpose, we performed EMSA and CHIP assay. Results showed the DNA binding activity of NF-κB was significantly increased in the cells transfected with pCMV-HBx detected by a biotin-labeled NF-κB1 binding oligonucleotides probe in the IP-10 promoter (-125 to-102) and a 177-bp DNA fragment in IP-10 promoter was amplified by PCR from DNA which isolated from cells transfected with pCMV-HBx and then immunoprecipitated with anti-p50 or anti-p65 antibody. These data suggested that the NF-κB directly binds to the NF-κB1 binding site in the IP-10 promoter.
To further defined the mechanism of HBx activated NF-κB activation. In this regard, by western blot, promoter luciferase activity and confocal assay we found after transfection of HBx plasmid into HepG2 cell line, the phosphorylation of TAK1 and IKKa was observed in HBx plasmid group. By addition TAK1 inhibitor or TAK1 siRNA, both NF-κB activity and IP-10 promoter activity were reduced. TRAF family member TRAF2 knockdown also impaired the NF-κB and IP-10 activity induced by HBx. Consistently, the inhibition of the nuclear translocation of NF-κB subunit p65 was defined after knockdown of TRAF2 or TAK1. Taken together, our data suggested that HBx-induced IP-10 expression might be mediated by TRAF2/TAK1/NF-κB signaling pathway.
4. HBx-induced IP-10 increases migration of peripheral blood leukocytes (PBLs)
We performed migration assay and found that PBLs exhibited decent increase of migratory activity in response to supernatants from cells transfected with plasmids expressing HBx, p50, or p65 comparing mock plasmids. The addition of neutralizing anti-IP-10 but not the control antibody to the supernatants significantly impaired this migration. Interestingly, NF-κB inhibitor SN50 treatment could decrease the chemotactic activity after transfection of pCMV-HBx. Together, these data suggested that HBx-induced IP-10 may mediate the migration of PBLs and NF-κB activity is involved in this process.
[Conclusions] Our study found that the mRNA and protein levels of IP-10 were much higher in HBV positive liver cancer tissues, compared to HBV negative liver cancer tissues or normal Hepatic hemangioma liver tissues, and may play an important role in HBV-induced inflammation of the liver injury. Then we transfected different HBV protein expressing plasmids into HepG2 cells and explored a novel molecular mechanism of HBV infection inducing IP-10 expression, which involves viral protein HBx affecting TRAF2/TAK1/NF-κB pathway, leading to the transactivation of IP-10 promoter. Our study provides insight into the migration of leucocytes in response to HBV infection, thus causing immune pathological injury of liver.
引文
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