锌对于原发性肝细胞癌BEL-7404细胞生物学行为的影响

目的:观察外源锌对原发性肝细胞癌(HCC)BEL-7404细胞生物学行为的影响。方法:应用TSQ锌离子荧光探针、MTT法、DNA倍体法、吖啶橙/溴乙啶双荧光染色法和Transwell小室法分别检测不同浓度硫酸锌刺激下BEL-7404细胞内锌离子含量、细胞活力、细胞周期、细胞凋亡及细胞迁移和侵袭能力的变化。应用real-time PCR和Western blot法分别检测不同浓度锌离子对BEL-7404细胞白蛋白的mRNA和蛋白表达的影响。结果:随着培养环境中锌离子浓度的升高,BEL-7404细胞内的锌离子含量增加,细胞的存活率和细胞迁移与侵袭能力降低,凋亡率升高(P0.05);G0/G1期细胞比例降低,G2/M期细胞比例升高(P0.05);细胞白蛋白的mRNA和蛋白表达量增加(P0.05)。结论:外源性给予锌离子可抑制HCC细胞的活力、迁移与侵袭能力,诱导细胞凋亡,阻滞细胞周期在G2/M期,并可能降低细胞的恶性表型。     

ZIP14对人肝细胞癌BEL-7404细胞生物学行为的影响

目的:研究锌转运蛋白ZRT/IRT样蛋白14(ZIP14)在原发性肝细胞癌(HCC)组织中的表达,以及ZIP14过表达对人肝细胞癌BEL7404细胞生物学行为的影响。方法:应用real-time PCR法与免疫组织化学法分别检测HCC组织与癌旁组织ZIP14的mRNA与蛋白表达。构建携带GV365-ZIP14的慢病毒表达体系,转染低表达ZIP14的BEL7404细胞,建立ZIP14过表达的细胞模型7404-ZIP14;应用MTT法、DNA倍体法和Transwell小室实验分别检测不同浓度硫酸锌刺激下感染GV365-ZIP14表达慢病毒的BEL7404细胞活力、细胞周期及迁移与侵袭能力的变化。结果:HCC组织ZIP14的mRNA表达水平和蛋白强阳性表达率均明显低于癌旁组织(P0.01);构建的GV365-ZIP14表达慢病毒感染HCC细胞株BEL7404使ZIP14过表达;与阴性对照组相比较,ZIP14过表达组细胞活力及迁移与侵袭能力明显下降,G_2/M期细胞比例明显升高,且变化的幅度随着培养环境锌离子浓度的提高而明显加大。结论:ZIP14在HCC组织呈低表达。ZIP14的过表达可抑制HCC细胞的活力、迁移能力和侵袭能力,并使细胞周期被阻滞在G_2/M期;其机制可能与ZIP14促进锌离子胞内转运而导致HCC细胞内锌积聚增多有关。     

肝细胞癌新型靶向基因治疗体系的构建及应用研究

构建肝细胞癌(HCC)新型靶向基因治疗体系,并以存活素(survivin)基因为靶点,探讨该体系体外杀伤肝癌细胞的作用。构建由巨细胞病毒(CMV)增强子和甲胎蛋白(AFP)启动子构成的融合启动子(AV)驱动的pcD-NA3.1(-)AV质粒,及含绿色荧光蛋白(GFP)的pcDNA3.1(-)AVGFP和含siRNA-survivin的pcDNA3.1(-)AV-siRNA-survivin质粒,以磷酸钙纳米为载体,导入HepG2、SMMC-7721和Hela细胞中,观察转染效率及体外对肝癌细胞的杀伤效应,并采用流式细胞技术(FCM)检测细胞凋亡率。结果显示:AV启动子可特异性驱动下游基因在HepG2细胞表达。此外,pcDNA3.1(-)AVsiRNA-survivin在HepG2细胞中可有效沉默survivin的mRNA和蛋白表达,并诱导68.8%的细胞死亡,而在Hela和SMMC-7721细胞中无显著作用。生长曲线结果显示,转染了pcD-NA3.1(-)AVsiRNA-survivin的HepG2细胞的生长显著受抑(P<0.05)。结果表明,该新型靶向基因治疗体系可高选择性作用于肝癌细胞,可为临床肝癌的基因治疗提供新的研究思路。     

miR-188-3p靶向YAP1抑制肝细胞癌的侵袭和迁移

目的 探究miR-188-3p在肝细胞癌(hepatocellular carcinoma,HCC)中的表达及其抑制HCC细胞侵袭和迁移的分子机制.方法 利用RT-qPCR检测miR-188-3p在人HCC组织和细胞系中的表达情况,进一步分析miR-188-3p表达在HCC中的临床价值;利用Western印迹检测转染m...     

Nucleophosmin/B23在肝细胞癌组织中的高表达

目的探讨Nucleophosmin／B23（B23）在肝细胞癌（HCC）组织中的表达及其临床病理意义。方法采用重组蛋白表达和杂交瘤细胞技术制备B23重组蛋白和鼠抗B23单克隆抗体。收集103例HCC组织、12例肝局灶性结节性增生和17例肝血管瘤旁肝组织的临床病理档案资料,10例HCC及癌旁肝新鲜组织,采用免疫组织化学（ABC）方法、逆转录聚合酶链反应（RT-PCR）及免疫印迹等技术检测B23在这些组织中的表达,并与增殖细胞核抗原（PCNA）的表达相比较。应用统计学方法对结果进行分析。结果RT-PCR及免疫印迹结果显示：在mRNA和蛋白质水平,B23在HCC组织中的表达明显高于对应的癌旁肝组织。免疫组织化学结果显示：B23在4组（HCC组、癌旁肝组织组、肝局灶性结节性增生组、血管瘤旁肝组织组）中的表达差异有统计学意义（P〈0．001）,其中B23在HCC组织中的表达显著高于其他3组（P〈0．01）,PCNA在HCC组织中的表达也显著高于其他3组（P〈0．01）,相关性分析表明B23与PCNA在4组中表达强度及其差异具有相关性（r=0．4767,P〈0．01）。B23在HCC组织中的表达强度与患者血清AFP水平、肿瘤病理分级及是否伴有肝硬化之间的关系有统计学意义（P〈0．05）。结论B23在HCC中呈高表达,且显著高于非癌肝组织;B23可作为HCC细胞增生程度的潜在标记,并在临床病理上具有潜在的应用意义。     

埃兹蛋白在肝细胞癌及其转移灶中的表达及意义

目的:了解人埃兹蛋白(ezrin)在正常肝、肝硬化组织、肝细胞癌(HCC)及其转移灶中的表达情况,并探讨其表达与肝细胞癌侵袭转移的关系。方法:用两步免疫组化染色法检测6例正常肝,25例肝细胞癌及14例转移灶中埃兹蛋白的表达;用Westernblot检测上述组织及12例肝硬化组织中埃兹蛋白的表达。结果:在正常肝、肝细胞癌及转移灶中,埃兹蛋白的强阳性表达率分别为0%、28%、57.1%,各组织中埃兹蛋白的表达差异均有统计学意义(P<0.05)。Westernblot的结果表明,转移灶中埃兹蛋白平均表达量是正常肝中的12.6倍,硬化肝中的4.7倍,肝细胞癌中的1.8倍。结论:埃兹蛋白的高表达与肝细胞癌的侵袭转移关系密切,可能是影响肝细胞癌患者预后及癌细胞转移的一个重要因素。     

食管癌相关基因4在肝细胞肝癌中的表达和功能

目的了解人食管癌相关基因4（ ECRG4）在人肝细胞肝癌组织和肝癌细胞系中的表达情况,探讨ECRG4对肝癌细胞增殖、凋亡以及迁移能力的影响。方法提取24例配对肝细胞肝癌组织和癌旁肝组织的总RNA以及正常肝细胞系QSG7701和肝癌细胞系HepG2的总RNA和总蛋白,分别利用即时荧光定量PCR（ RT-qPCR）和Western blot法检测ECRG4的表达;构建ECRG4的真核表达质粒ECRG4-pcDNA3.1,转染肝癌细胞系HepG2,进行体外的细胞增殖、凋亡以及迁移实验。结果与癌旁肝组织相比,在肝细胞肝癌组织中ECRG4 mRNA表达下调或缺失（98.5％,23／24,P＜0.01）;和正常肝细胞系QSG7701相比,ECRG4 mRNA在肝癌细胞系HepG2中的表达明显下调（ P＜0.05）并且其蛋白表达水平也明显降低。转染ECRG4表达质粒后HepG2细胞的增殖和迁移能力明显低于对照组（P＜0.05）,细胞凋亡率明显高于对照组（P＜0.05）。结论 ECRG4在肝细胞肝癌中表达下调,在体外肝癌细胞系中过表达ECRG4可抑制细胞的增殖和迁移,促进细胞凋亡,ECRG4可能是肝细胞肝癌潜在的抑癌基因,有望为肝细胞肝癌分子靶向治疗提供新策略。     

E-cadherin的表达与肝细胞癌侵袭和转移的关系

目的研究上皮性钙黏蛋白（E-cadherin）在人肝细胞癌（HCC）中的表达情况,探讨其与肝癌的相关性。方法选择56例有完整随访资料的肝细胞癌及相应癌旁组织标本、20例正常肝组织标本,用RT-PCR方法检测E-cadherinmRNA的表达;用免疫组织化学方法检测E-cadherin的表达。结果①E-cadherin在肝细胞癌组织中的表达显著低于癌旁组织和正常组织（P〈0．05）,而在癌旁组织和正常组织中的表达无差异;②E-cadherin在肝癌组织中的表达与术后复发时间呈正相关（P〈0．05）,与病理分期呈负相关（P〈0．05）;③癌组织中E-cadherin表达与肝外转移呈负相关（P〈0．05）;④癌旁组织中E-cadherin表达与术后复发时间呈正相关（P〈0．05）。结论E-cadherin表达缺失或下调与肝癌的分化程度、侵袭转移能力和复发倾向相关,对肝癌临床转归的评估有一定指导意义。     

肝细胞癌中HP1BP3的表达及其对细胞恶性生物学行为的影响

目的 检测分析异染色质蛋白1结合蛋白3(heterochromatin protein 1 binding protein 3, HP1BP3)在肝细胞癌中的表达及其对细胞恶性生物学行为的影响。方法 应用TCGA数据库分析肝细胞癌与癌旁组织中HP1BP3基因的表达差异。生存检验分析HP1BP3表达与肝细胞癌患者总生存期的关系,单因素及多因素分析相关危险因素。应用Western blot和免疫组化法检测HP1BP3蛋白表达水平。用小干扰RNA(siRNA)分别转染Huh7、HepG2细胞,功能实验检测细胞增殖、迁移和侵袭能力。通过GSEA分析对其作用机制进行探究。结果 TCGA数据库分析结果显示,HP1BP3基因在肝细胞癌组织中的表达明显高于非肿瘤组织(P<0.01),高表达组患者的总生存期较低表达组短(P=0.029)。HP1BP3基因是肝细胞癌的独立预测因子(P=0.024)。体外实验结果显示,HP1BP3基因在肝癌细胞株及肝细胞癌组织中表达上调。沉默HP1BP3基因可抑制肝癌细胞的增殖、侵袭和迁移。HP1BP3高表达与泛素化(P<0.001)、核酸切除修复(P<0...     

肝细胞癌中DcR3表达与癌细胞凋亡的关系研究

目的探讨诱捕受体3（decoy receptor 3,DcR3）蛋白在原发性肝细胞癌（HCC）中的表达及其与癌细胞凋亡和HCC预后的关系。方法应用免疫组织化学（EnVision法）及和脱氧核糖核酸末端转移酶介导的缺口末端标记（TUNEL）技术检测43例HCC,16例非癌肝组织（单纯性肝硬化及肝血管瘤周围正常肝组织）中DcR3蛋白的表达及凋亡情况,并分析其与临床病理参数的关系。结果DcR3明确定位于肝细胞胞质内;HCC组织中的DcR3蛋白的阳性率为74．42％（32／43）,明显高于非癌肝组织43．75％（7／16,P〈0．05）;HCC中有转移癌组DcR3阳性率为100％（22／22）,明显高于无转移组52．94％（9／17,P〈0．01）;DcR3蛋白的表达与AFP水平（r=0．444,P〈0．01）、门静脉癌栓（r=0．414,P〈0．01）有关,与年龄、性别、有无肝硬化、包膜浸润、肿瘤结节数及分化程度无关（P〉0．05）。HCC中细胞凋亡指数[AI（0．78±0．64）％]明显低于非癌肝组织[（3．32±1,81）％,P〈0．01];HCC临床TNM分期Ⅰ、Ⅱ期AI（1．03±0．69）％高于Ⅲ、Ⅳ期[（0．52±0．48）％,P〈0．01];HCC无转移组AI（1．10±0．72）％高于转移组[（0．44±0．27）％,P〈0．01];AI与AFP水平（r=-0．468,P〈0．01）、门静脉癌栓（r=-0．434,P〈0．01）、包膜浸润（r=-0,331,P〈0．05）有关,与年龄、性别、有无肝硬化、肿瘤结节数及分化程度无关（P〉0．05）。在HCC和非癌肝组织中,DcR3阳性者的AI均分别低于阴性者的AI（均P〈0．01）。结论DcR3表达可影响凋亡并在HCC的发生发展中起重要作用;检测DcR3蛋白与AI有助于判断HCC患者预后。     

HCA587 antigen expression in normal tissues and cancers: correlation with tumor differentiation in hepatocellular carcinoma

The HCA587 gene, identified by serological analysis of recombinant cDNA expression library (SEREX) from a hepatocellular carcinoma (HCC) patient, encodes a new member of cancer-testis antigens. HCA587 mRNA expression in normal tissues and cancers has been previously reported. To estimate its immunogenicity to induce immune response, it is essential to analyze HCA587 expression at the protein level. In this study anti-HCA587 polyclonal antibody, termed "TC-1," was generated, and the expression of HCA587 protein was assessed by immunohistochemical staining in a panel of normal and tumor tissue sections. No HCA587 protein was shown in normal tissues except germ cells in testis and Purkinji cells in cerebellum. In HCC specimens the HCA587 protein was expressed in 37.1% (26 of 70) samples. The expressed protein was either located in the cytoplasm or nucleus depending on the individual samples. More importantly, there appears to be correlation between the tumor differentiation of HCC and HCA587 protein expression, ie, the lower differentiation, the higher percentage of protein expression. Coincidentally, seroreactivity showed that the Ab specific to recombinant HCA587 protein was detected only in the sera of three patients with poorly differentiated HCCs. HCA587 antigen was also expressed in different proportions in melanoma, lymphoma, pancreatic cancer, and lung cancer.     

Identification of a new HLA-A*0201-restricted CD8+ T cell epitope from hepatocellular carcinoma-associated antigen HCA587

For the development of peptide-based cancer immunotherapies, we aimed to identify specific HLA-A*0201-restricted CTL epitopes in hepatocellular carcinoma (HCC) associated antigen HCA587, which has been identified as a member of the cancer/testis (CT) antigens highly expressed in HCC. We first combined the use of an HLA-A*0201/peptide binding algorithm and T2 binding assays with the induction of specific CD8(+) T cell lines from normal donors by in vitro priming with high-affinity peptides, then IFN-gamma release and cytotoxicity assays were employed to identify the specific HLA-A*0201 CD8(+) T cell epitope using peptide-loaded T2 cells or the HCA587 protein(+) HCC cell line HepG2. In the six candidate synthesized peptides, two peptides showed higher binding ability in T2 binding assays. No. 2 peptide, encompassing amino acid residues FLAKLNNTV (HCA587(317-325)), was able to activate a HCA587-specific CD8(+) T-cell response in human lymphocyte cultures from two normal donors and two HCC patients, and these HCA587-specific CD8(+) T cells recognized peptide-pulsed T2 cells as well as the HCA587 protein(+) HCC cell line HepG2 in IFN-gamma release and cytotoxicity assays. The results indicate that no. 2 peptide is a new HLA-A*0201-restricted CTL epitope capable of inducing HCA587-specific CTLs. Our data suggest that identification of this new HCA587/HLA-A*0201 peptide FLAKLNNTV may facilitate the design of peptide-based immunotherapies for the treatment of HCA587-bearing HCC patients.     

Elicitation of both CD4 and CD8 T-cell-mediated specific immune responses to HCA587 protein by autologous dendritic cells

We recently cloned a new member of cancer/testis antigen named HCA587, which was highly expressed in human hepatocellular carcinoma (HCC) tissues. To investigate it as a potential tumour-specific target for immunotherapy, the immunogenicity of this protein, especially the ability to induce specific cellular immune responses, was evaluated in the present study. As dendritic cells (DC) are the most potent antigen-presenting cells, DC-based vaccination has recently shown marked promise for the treatment of human malignancies by immunological intervention. Here, we demonstrate that autologous DC loaded with HCA587 protein could induce specific T-cell responses in healthy individuals by in vitro stimulations. Enzyme-linked immunospot analysis for interferon-gamma (IFN-gamma) secretion demonstrated HCA587-specific CD8(+) T cells in the antigen-stimulated peripheral blood lymphocytes, and the analysis of CD4(+) T cells by proliferation assay also showed antigen-specific reactivities in normal donors. Two-colour flow cytometric analysis of surface markers and intracellular cytokine expression demonstrated that HCA587-specific cytotoxic T lymphocytes exhibited a heterogeneous CD8(+)/CD56(+) expression, and a striking T-helper 1 cytokine bias (IFN-gamma(high)/IL-4(low)) was observed for both CD4(+) and CD8(+) HCA587-specific lymphocyte populations. We conclude that HCA587 is a potent immunogen that can induce CD4(+) and CD8(+) T-cell-mediated specific immune responses, and these findings propose HCA587 as a good candidate for the development of a therapeutic protein-based DC tumour vaccine for the treatment of HCC patients.     

Down-regulation of cancer/testis antigen OY-TES-1 attenuates malignant behaviors of hepatocellular carcinoma cells in vitro

Cancer/testis (CT) antigens are normally expressed in testis and overexpressed in various tumor types. However, their biological function is largely unknown. OY-TES-1, one of cancer/testis (CT) antigens, is reported overexpression in hepatocellular carcinoma (HCC). And we assumed that OY-TES-1 contribute to oncogenesis and progression of HCC. In this study, we knocked down OY-TES-1 by small interference RNA (siRNA) in HCC cell lines (HepG2 and BEL-7404) to verify this assumption and evaluate its potential as therapeutic targets for HCC. We showed that down regulation of OY-TES-1 decreased cell growth, induced the G₀/G₁ arrest and apoptosis, and prevented migration and invasion in the two HCC cell lines. Further analysis revealed that down regulation of OY-TES-1 increased expression of apoptosis-regulated protein caspase-3, and decreased expression of cell cycle-regulated protein cyclin E, migration/invasion-regulated proteins MMP2 and MMP9. These findings may shed light on the gene therapy about the OY-TES-1 expression in HCC cells.     

肝癌细胞特异性内在化肽的筛选和初步鉴定

应用噬菌体肽库技术筛选与肝癌细胞特异性结合并可以内在化的短肽,为肝癌的导向治疗奠定基础。以肝癌细胞株BEL-7404作为筛选靶细胞,对噬菌体展示随机12-肽库进行亲和淘选。经过3轮淘选后,共随机挑选出20个噬菌斑进行扩增和测序,同时用细胞ELISA检测其与肝癌细胞的结合情况,并通过免疫荧光术、流式细胞术等方法进一步鉴定噬菌体克隆对肝癌细胞的导向性及内在化的效果。结果表明3轮淘选所得的噬菌体回收率逐步提高,显示淘选过程对随机肽库有一定的富集效果。从扩增的噬菌体克隆中选择结合力最强的LJ08通过免疫荧光术、流式细胞术鉴定,结果显示LJ08与肝癌细胞呈特异性结合并内在化进入肝癌细胞。结论认为,通过对噬菌体随机肽库的筛选,得到可以与肝癌细胞BEL-7404结合并可以内在化的噬菌体肽,为该肽作为肝癌导向治疗的药物载体奠定基础。     

Up-regulation of long non-coding RNA Sox2ot promotes hepatocellular carcinoma cell metastasis and correlates with poor prognosis

Background: Long non-coding RNAs (lncRNAs) have been shown to have important regulatory roles in cancer biology, and the lncRNA Sox2ot is up-regulated in some tumors. However, the contributions of Sox2ot to hepatocellular carcinoma (HCC) remain largely unknown. Methods: In the present study, expression of lncRNA Sox2ot was evaluated by quantitative real-time PCR in tumor tissues and adjacent non-tumor tissues in 84 HCC patients. The association of lncRNA Sox2ot expression with clinicopathological features and the prognosis of HCC patients were also analyzed. Survival analysis was performed using the Kaplan-Meier method and Cox’s proportional hazards model. Small interfering RNA assay was used to explore the function of lncRNA Sox2ot on HCC cell migration and invasion. Results: lncRNA Sox2ot expression level was significantly higher in HCC tissues compared with adjacent non-tumor tissues (P<0.05). High expression of lncRNA Sox2ot was associated with histological grade, TNM stage, and vein invasion. The 5-year overall survival of high lncRNA Sox2ot expression group was significantly shorter than that of low lncRNA Sox2ot expression group (P<0.05). The multivariate Cox regression analysis indicated that lncRNA Sox2ot expression was an independent prognostic factor for overall survival. In addition, the metastasis ability of HCC cells was significantly decreased by knocking down lncRNA Sox2ot expression. Conclusions: The results suggested that lncRNA Sox2ot played crucial roles in promoting HCC cell migration and invasion, and might represent a novel prognostic biomarker for HCC.     

syndecan-1表达对肝细胞癌增殖、血管生成和转移的影响

目的探讨syndecan-1在肝细胞癌（hepatocellular carcinoma,HCC）表达的临床病理意义,及其与肿瘤转移、增殖和血管生成的关系。方法用免疫组织化学染色检测了syndecan-1在30例HCC原发癌组织、癌旁组织和肝内转移灶的表达,血管内皮生长因子（vascularendothelial growth factor,VEGF）和CD34-微血管密度（microvessel density,MVD）在原发灶和肝内转移灶的表达,及Ki-67在原发灶的表达。结果与HCC癌旁组织相比,syndecan-1在原发癌组织表达水平明显增高,syndecan-1在肝内转移灶表达水平明显下降（χ2=25.62,P〈0.001）。在HCC原发癌组织中,syndecan-1表达强度与Ki-67指数呈正相关（r=0.386,P〈0.05）。在肝内转移灶中,syndecan-1表达强度与CD34-MVD呈正相关（r=0.370,P〈0.05）。结论 HCC组织syndecan-1表达可能影响肿瘤转移、增殖和血管生成,提示syndecan-1在HCC生长和转移中的特殊作用可能为HCC有效治疗策略提供新思路。     

蛋白激酶CβⅡ诱导上皮-间质转化及血管新生促进肝癌发展

目的 探讨蛋白激酶CβⅡ（PKCβⅡ）在肝细胞癌(HCC)发展中的作用机制。方法 免疫印迹法观察PKCβⅡ在肝细胞系L02和肝癌细胞系SK-hep1、HepG2、BEL-7404、7721、Hep3B和huh7中的表达，构建稳定高表达PKCβⅡ的细胞系，倒置相差显微镜下观察细胞形态变化，免疫荧光观察E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）表达的变化；通过免疫印迹法、实时定量聚合酶链反应(Real-time PCR)和放线菌酮（CHX）追踪实验，观察PKCβⅡ调控E-cadherin、N-cadherin和Snail表达的分子机制；小室迁移和侵袭实验(transwell assay)以及裸鼠尾静脉注射观察PKCβⅡ对肝癌细胞转移的影响；成管实验观察PKCβⅡ对人脐静脉内皮细胞（HUVECs）血管形成能力的影响，酶联免疫吸附法（ELISA）观察PKCβⅡ对肝癌细胞上清中血管内皮生长因子A（VEGFA）含量的影响。结果 PKCβⅡ在肝癌细胞系中的表达高于肝细胞系L02，PKCβⅡ促进肝癌细胞形态从鹅卵石样上皮细胞向梭行间质样细胞的转变，通过mRNA水平下调E-cadherin（P<0.05）和上调N-cadherin（P<0.01）的蛋白表达，通过翻译水平上调Snail蛋白表达，PKCβⅡ还促进了肝癌细胞的迁移、侵袭（P<0.01）以及VEGFA的分泌（P<0.01）和血管新生（P<0.01）。结论 蛋白激酶CβⅡ诱导上皮-间质转化及血管新生在肝癌的发展中有重要作用。