Abnormal β-catenin gene expression with invasiveness of primary hepatocellular carcinoma in China

AIM To study the abnormal expression of β-catenin gene and its relationship with invasiveness of primary hepatocellular carcinoma among Chinese people. METHODS Thirty-four hepatocellular carcinoma (HCC) specimens and adjacent para-cancerous tissues, 4 normal liver tissues were immunohistochemically stained to study subcellular distribution of β-catenin. Semiquantitive analysis of expression of β-catenin gene exon 3 mRNA was examined by RT-PCR and in situ hybridization. The relationship between expressions of both β-catenin protein, mRNA and clinicopathological characteristics of HCC was also analyzed. RESULTS Immunohistochemistry showed that all normal liver tissues and para-cancerous tissues examined displayed membranous type staining for β-catenin protein,occasionally with weak expression in the cytoplasm.While 21 cases (61.8%) of HCC examined showed accumulated type in cytoplasms or nuclei. The accumuled type Labling Index (LI) of cancer tissue and paracancarous tissue was (59.9 ± 26.3) and (18.3 ± 9.7)respectively (P＜0.01). Higher accumulated type LI was closely related with invasiveness of HCC. Results of RTPCR showed the β-catenin gene exon 3 mRNA Expression Index (El) of 34 HCCs was higher than that of paracancerous tissue and normal liver tissue. Using in situ hybridization, the signal corresponding to β-catenin gene exon 3 mRNA was particularly strong in cytoplasm of HCC when compared with those of para-cancerous and normal liver tissues. Over expression of β-catenin exon 3 was also found to be correlated with high metastatic potential of HCC. CONCLUSION Abnormal expression of β-catenin gene may contribute importantly to the invasiveness of HCC among Chinese people.     

Mutation and overexpression of the beta-catenin gene may play an important role in primary hepatocellular carcinoma among Chinese people

AIM: To study the role of beta-catenin gene mutation and expression in hepatocellular carcinogenesis. METHOD: Thirty-four hepatocellular carcinoma (HCC) specimens and adjacent para-cancerous tissues, and four normal liver tissues were analyzed. Subcellular distribution of beta-catenin was examined by immunohistochemistry staining. Mutation and semiquantitative expression of beta-catenin gene exon 3 mRNA were detected by RT-PCR-SSCP and in situ hybridization. RESULT: Immunohistochemistry showed that all normal liver tissues and para-cancerous tissues examined showed membranous-type staining for beta-catenin protein, frequently with weak expression in the cytoplasm, but no beta-catenin accumulation in nuclei was found; while in liver cancer, 21 cases (61.8%) of HCC examined showed accumulated type in cytoplasms or nuclei. On SSCP, 15 cases (44.1%) of HCC altogether displayed three kinds of characteristic mutational mobility shifts. No abnormal shifting bands were found in tissues from normal liver or para-cancerous area. The beta-catenin gene exon 3 mRNA expression index of 34 HCCs was higher than that of para-cancerous tissue and normal liver tissue. Using in situ hybridization, the signal corresponding to beta-catenin gene exon 3 mRNA was particularly strong in cytoplasm of HCC when compared with those of paracancerous tissues and normal liver tissues. CONCLUSION: beta-catenin gene mutation and overexpression may have a critical role in malignant progression of hepatic carcinogenesis among Chinese people.     

Inhibition mechanism of a newly cloned candidate tumor suppressor gene JST during hepatocarcinogenesis and its abnormal expression in human hepatocellular carcinoma from Qidong liver cancer risk area, China

BACKGROUND/AIMS: To study inhibition effect of a newly cloned candidate tumor suppressor gene (JST) during hepatocarcinogenesis and its normal expression in human hepatocellular carcinoma from Qidong liver cancer risk area, China. METHODOLOGY: By preparing rabbit anti-human JST polyclonal antibody, constructing of JST frameshift mutant and exploring RT-PCR, in situ hybridization, immunohistochemistry, Western blot, Northern blot, cDNA expression microarray, co-immunoprecipitation and the tumorigenicity assay in vivo and in vitro, gene treatment, etc, JST gene expression and inhibition tumor growth effects were analyzed, including 150 pairs of HCC specimens and their adjacent para-cancerous tissues, 8 cases of normal liver tissues and QGY7701, HepG2, Hep3B cell line. Its relationship with the invasiveness of HCC from Qidong was also investigated. RESULTS: Our results showed that there is expression difference for JST between liver cancer and para-cancerous tissue and the results of RT-PCR, in situ hybridization, immunohistochemistry, Western blot, Northern blot suggested that it is a down-regulation gene. The labeling index (LI) of cancer tissue and para-cancerous tissue was (70.2+/-8.7) and (9.4+/-2.8) respectively (p<0.01), lower LI was closely related with invasiveness of HCC, decreased expression of JST was also shown by Western blotting. Results of RT-PCR showed the JST gene expression index (EI) of HCCs was lower than that of para-cancerous tissue and normal liver tissue and there are some sequence differences between cancer and para-cancerous tissues. Northern blot showed JST having down-regulation expression among 92.20% (138/150) of patients. Using in situ hybridization, the signal corresponding to JST mRNA was particularly weak in cytoplasm of HCC when compared with those of para-cancerous and normal liver tissues. Less expression of JST was also found to be correlated with high metastasis potentiality of HCC. JST overexpression inhibits DNA synthesis and apoptosis in QGY7701 cells. QGY7701 cell transfected with JST is more inhibited in soft agar than that of vector transfected control cells (p<0.01) or QGY7701 cells stably transfected with the JST frameshift mutant. The tumor formation is more inhibited in the QGY7701-pcDNA3.1-JST group than that in the QGY7701-pcDNA3.1, QGY7701-pcDNA3.1-JST frameshift mutant group. cDNA expression microarray showed expression differences of 10% (20/200,18 up-regulation; 2 up-regulation) tumor genes were considered significant between QGY7701-pcDNA3.1-JST and QGY7701-pcDNA3.1. Using a co-immunoprecipitation approach, intracellular binding of JST and p53 was found. Higher levels of p53 were detected following infection with pcDNA3.1-JST when compared with pcDNA3.1. Induction of FasL could be demonstrated in Hep3B and in HepG2 cells following transfection pcDNA3.1-JST, but not following transfection pcDNA3.1. CONCLUSIONS: JST is a putative tumor suppressor gene. Overexpression of JST gene may contribute to inhibiting the occurrence, advancement and invasiveness of HCC from Qidong, a high risk area in China.     

用cDNA阵列技术研究黄曲霉毒素B1诱发树鼩肝癌形成过程中的基因变化

目的 对树鼩肝癌形成过程中的基因表达差异进行动态分析,探讨肝癌发生的分子机制。 方法用cDNA阵列技术,将2例黄曲霉毒素B1诱发的树鼩肝癌组织分别与其癌旁组织和其肝癌形成前的活检肝组织、实验前对照和同期对照肝组织进行基因表达水平的6种比较分析。结果 不同的比较方式所显示的差异表达的基因谱不同,可归为4类:癌组织表达高于癌旁组织、癌旁组织表达高于癌发生前肝的组织;癌与癌旁表达水平相仿,但高于癌发生前的肝组织;癌组织下调,低于癌旁组织;癌发生前表达上调,在癌发生后表达下调。 结论 对肝癌形成过程中不同时期的肝组织基因表达水平进行动态对比分析,有助于阐明肝癌发生的分子机制并最终筛选出与肝癌发生有关的关键基因。     

肝癌细胞表达差异片段MRG 98.2的临床意义

目的 筛选肝细胞癌（HCC）相关基因,探讨其在HCC发病中的临床意义。方法 用差异显示技术对比研究正常肝组织、HCC组织及癌旁肝组织之间mRNA的表达差异,以肝cDNA片段MRG98．2为探针,对10例HCC及癌旁肝组织进行斑点印渍分析,并通过逆转录聚合酶链反应（RT－PCR）方法检测这些标本血管内皮生长因子（VEGF）mRNA的表达水平。结果 从HCC组织中获得的差示片段MRG98．2在7例HCC标本中表达阳性（70％）,癌旁肝组织弱表达（2／10）或无表达。VEGF mRNA在7例MRG98．2 阳性表达的HCC组织中的6例表达上调。结论 MRG98．2是HCC相关的基因片段,其表达与VEGF mRNA相关,并可能与肿瘤浸润转移、患者的预后不良有关。     

Tissue microarray for high-throughput analysis of gene expression profiles in hepatocellular carcinoma

AIM: To study the expression profiles of HBsAg, HBcAg, p21WAF1/CIP1 (p21), Rb genes in hepatocellular carcinoma (HCC) and to investigate their roles in the hepatocar-cinogenesis. METHODS: HCC tissue microarray containing 120-min tissues of 40 HCC cases was constructed. HBsAg, HBcAg, p21 and Rb proteins were immunohistochemically stained by streptavidin-peroxidase conjugated method (S-P). The expression loss of these genes in cancerous, para-cancerous tissues and adjacent normal liver tissues of 40 HCCs were comparatively examined. RESULTS: The positive rate of HBsAg expression in cancerous tissues of 40 HCCs was 7.5%, which was lower than that in para-cancerous and adjacent normal liver tissues (X2=12.774, P<0.01; X2=18.442, P<0.01). The positive rate of HBcAg expression in cancerous tissues of 40 HCCs was 20.0%, which was also lower than that in para-cancerous and adjacent normal liver tissues (X2=9.482, P<0.01; X2=14.645, P<0.01). p21 protein deletion rate in cancerous tissues of 40 HCCs was 27.5%, which was higher than that in para-cancerous and adjacent normal liver tissues (X2=7.439, P<0.01; X2=11.174, P<0.01). p21 protein deletion correlated remarkably with the pathological grade of HCC (X2=0.072, P<0.05). Rb protein deletion rate in cancerous tissues of 40 HCCs was 42.5%, which was also higher than that in para-cancerous and adjacent normal liver tissues (X2=10.551, P<0.01; X2=18.353, P<0.01). Rb protein deletion rate did not correlate remarkably with tumor size or pathological grade of HCC (X2=0.014, P>0.05; X2=0.017, P>0.05). CONCLUSION: Expression deletion of HBsAg, HBcAg, p21 and Rb proteins in HCCs may play important roles in the carcinogenesis of HCC. Tissue microarray is an effective high-throughput technique platform for cancer research.     

乙型肝炎病毒相关性肝癌组织维生素D受体表达情况分析

目的 探讨乙型肝炎病毒相关性肝细胞癌（HCC）组织维生素D受体（VDR）表达变化及其与癌旁肝硬化组织和正常组织的异同。 方法 取10例乙型肝炎病毒相关性HCC组织及其癌旁肝组织和4例正常肝组织,采用实时荧光定量PCR法检测组织VDR mRNA水平,采用免疫组织化学法检测组织VDR表达,采用Western blot法检测组织VDR蛋白表达。 结果 免疫组织化学法检测发现HCC组织、癌旁组织和正常肝组织均有VDR表达,主要在细胞质表达,而细胞核基本无表达;癌组织VDR mRNA为（2.77±0.30）,显著高于癌旁肝硬化组织的（1.62±0.21）或正常肝组织的【（1.57±0.19）,P<0.01】;Western blot分析发现,HCC组织VDR蛋白相对表达量为（1.15±0.57）,显著高于癌旁肝硬化组织的（1.02±0.25）或正常肝脏组织的【（0.37±0.11）,P<0.01】。 结论 VDR可能参与了HCC的发生发展过程。     

DLC-1基因在肝细胞癌组织中表达的研究

目的 研究肝癌缺失基因-1（DLC-1）在人原发性肝癌组织中的表达及与肝细胞癌侵袭转移的关系。方法收集51例复旦大学附属中山医院外科手术切除的肝细胞癌及癌旁正常组织标本，用荧光定量RT-PCR方法分析组织中DLC-1基因的表达。结果在51对配对的肝细胞癌与癌旁无瘤肝组织中，前者DLC-1基因表达水平明显低于后者（P〈0．01），且高侵袭性肝细胞癌组明显低于低侵袭性肝细胞癌组（P〈0．05）。结论DLC-1基因在HCC组织中呈低表达，其表达水平与其侵袭性高低相关。     

γ-谷氨酰转移酶mRNA亚型对肝细胞癌变的监测

目的 探讨γ-谷氨酰转移酶（GGT）mRNA亚型的转化与原发性肝癌（HCC）发生的关系,寻找肝癌早期诊断的新方法。方法 以逆转录聚合酶链反应方法检测正常对照组、非癌肝病组、肝癌组及肝转移癌肝组织及外周血的3种GGTmRNA亚型(A、B、C亚型)。结果 正常肝纤维、非癌肝病的肝组织及肝转移癌癌周组织主要的GGTmRNA类型为A亚型,肝癌组织、癌旁组织及远癌组织GGTmRNA-B亚型的阳性率显著高于正常肝脏及非癌肝癌的肝组织（P＜0．05）,癌组织GGT mRNA-A亚型阳性率明显低于正常对照及非癌肝病组（P＜0．05）。在26例HCC中有12例外周血中检出GGTmRNA-B亚型,甲胎蛋白阴性的10例HCC中有5例检出GGTmRNA-B亚型。结论 GGT mRNA亚型转化与肝癌发生有密切关系,分析GGT基因可望成为监测肝细胞癌变的灵敏方法。     

Expression of β-catenin in hepatocellular carcinoma

AIM: The β-catenin has been recognized as a critical member of the Wnt signaling pathway and plays an important role in the generation/differentiation of many tissues. Inappropriate activation of this pathway has been implicated in carcinogenesis. The mechanism underlying the development as well as its prognosis of hepatocellular carcinoma (HCC) has remained unclear. The purpose of this study is to analyze the expression of β-catenin in HCC in relation to histological grades and viral hepatitis backgrounds. METHODS: Thirty-two sections were selected at random from autopsy and surgical cases of HCC. Immuohistologically, the location and positivity of β-catenin expression in HCC was examined. RESULTS: Normal hepatocytes did not express β-catenin. In 78% of HCC β-catenin was expressed at the membrane of the cells, with or without cytoplasmic and/or nuclear expression. The tumor cells with well- and moderately-differentiated grades expressed frequently at the membrane and cytoplasm compared with poorly-differentiated type. Nuclear expression of β-catenin was prone to occur in the tumor cells of poorly-differentiated grade. There were 15% of hepatitis C virus (HCV) backgrounds with nuclear expression. In contrast, there was 38% with nuclear expression in hepatitis B virus (HBV) backgrounds. In nonBnonC hepatitis, no case expressed nuclear β-catenin. CONCLUSION: The β-catenin expression in HCC cells was heterogenous among types of hepatitis viral infection. Wnt signaling pathway might be deeply involved in less-differentiated HCC and HBV background.     

Cloning and expression of MXR7 gene in human HCC tissue

AIM To clone and identify the whole cDNA of MXR7 gene and to find out its expression in human HCC, and normal tissues.METHODS The DNA primers were designed and synthesized according to the whole cDNA sequence of MXR7 gene. The cDNA of human HCC was taken as the template while the cDNA of MXR7 gene was synthesized by polymerase chain reaction (PCR). Recombinant DNA conforming to reading frame was constructed by connecting purified PCR product of the cDNA of MXR7 gene with expression vector pGEX-5X-1 of fusion protein. The plasmid MXR7/pGEX-5X-1 was identified by sequencing. Using 32P labeled MXR7 cDNA as probe, MXR7 mRNA expression was detected by Northern blot analysis in 12 different human normal tissues, 7 preoperatively untreated non-liver tumor tissues, 30 preoperatively untreated HCC, the paracancerous liver tissues and 12 normal liver tissues samples.RESULTS Restriction enzyme and sequence analysis confirmed that the insertion sequence in vector pGEX-5X-1 was the same as the cDNA sequence of MXR7 gene. Northern blot analysis showed no expression of MXR7 mRNA in 12 kinds of normal human tissues including liver, 7 tumor tissues in other sites and 12 normal liver tissues, the frequencies of MXR7 mRNA expression in HCC and paracancerous liver tissues were 76.6% and 13.3%, respectively.The frequency of MXR7 mRNA expression in HCC without elevation of serum AFP and in HCC ＜5cm was 90% (9/10) and 83.3% (5/6),respectively.CONCLUSION MXR7 mRNA is highly expressed in human HCC, which is specific and occurs at an early stage of HCC, suggesting MXR7 mRNA can be a tumor biomarker for HCC. The detection of MXR7 mRNA expression in the biopsied liver tissue is helpful in discovering early subclinical liver cancer in those with negative serum AFP.     

Cartilage oligomeric matrix protein expression in hepatocellular carcinoma and the cirrhotic liver

BACKGROUND: Cartilage oligomeric matrix protein (COMP) is the fifth member of the thrombospondin family of extracellular, calcium-binding proteins. It was initially isolated and characterized in cartilage tissue, where it is thought to contribute to the extracellular matrix composition and cell-extracellular matrix interaction. In the present study the expression of COMP was investigated in normal liver (n=19), liver cirrhosis (n=14) and hepatocellular carcinoma (HCC; n=16) tissues, both at the mRNA and protein level. METHODS AND RESULTS: By northern blot and western blot analysis, COMP was absent or rarely expressed in the normal liver and liver cirrhosis tissues, but significantly overexpressed in HCC tissue samples. The COMP mRNA overexpression in HCC was not related to the clinical stage or tumor grade. By in situ hybridization and immunohistochemistry analysis, COMP mRNA and protein expression were localized within the cytoplasm of the tumor cells. CONCLUSION: COMP is highly expressed within the tumor cells of HCC, suggesting that COMP might play a role in the pathophysiology of this disease.     

PTEN和磷酸化Smad2在原发性肝癌中的表达

目的探讨肝癌等组织中10号染色体缺失的磷酸酶及张力蛋白同源物（PTEN）和磷酸化Smad2（P-Smad2）表达的意义。方法采用免疫组织化学技术检测肝癌组织、癌旁肝组织和非癌性肝组织中P-Smad2和PTEN的表达。结果31份肝癌组织中PTEN表达以细胞质和细胞膜明显,细胞核基本不表达；25份癌旁及13份非癌性肝组织中则以细胞核和细胞质强表达,细胞膜弱表达。PTEN在肝癌组织的表达率（64．5％）低于癌旁肝组织（96．0％）和非癌性肝组织（100．0％）,表达强度（4．19±3．31）低于癌旁肝组织（7．88±0．93）和非癌性肝组织（7．77±0．93）,差异均有统计学意义（P＜0．05）。不同病理分级的肝癌组织中PTEN的表达率差异无统计学意义（P＞0．05）,≥Ⅱ级的肝癌组织细胞质表达强度（3．07±2．87）低于＜Ⅱ级（5．80±3．12）的肝癌组织（P＜0．05）。癌旁有、无肝内血管癌栓的肝癌组织中,PTEN表达率分别为45．5％和85．7％,表达强度分别为3．00±3．46和6．28±2．37,差异均有统计学意义（P＜0．05）。PTEN在肝细胞的表达定位与病理类型呈负相关（r=0．34,P＜0．01）,与肝内血管癌栓呈负相关（r=-0．43,P＜0．05）。非癌性肝组织、癌旁肝组织和病理分级＜Ⅱ级的肝癌组织中,P-Smad2表达以细胞核和细胞质明显,≥Ⅱ级的肝癌组织中则以细胞核为主。P-Smad2在肝细胞的表达定位与病理类型呈正相关（r=0．22,P＜0．05）,P-Smad2在细胞核的表达强度。肝癌组织与癌旁肝组织的差异无统计学意义,也与肝内血管有无癌栓无关。肝癌组织中PTEN和P-Smad2表达呈负相关（r=-0．73,P＜0．01）。结论PTEN的表达、强度以及和P-Smad2的核、质转位可能与肿瘤的发展和恶化有关,二者可能存在相互作用,共同参与肝癌的发生机制。     

肝癌组织中p27蛋白表达与肿瘤增殖凋亡活性的研究

研究肝细胞癌组织中p27表达对细胞增殖与凋亡活性的影响.采用Elivision法检测47例肝癌及相应癌旁肝组织中p27及PCNA的表达,TUNEL法检测原位细胞凋亡.结果显示肝癌组织中细胞增殖与凋亡指数明显高于癌旁肝组织.肝癌组织中p27染色指数明显低于癌旁肝组织与正常肝组织.低分化肝癌组织中p27表达和凋亡指数均明显降低.存在肝外转移的肝癌组织中p27表达降低而增殖指数明显增高.进一步研究显示,p27高表达组肝癌组织中凋亡指数明显高于低表达组,而增殖指数却显著降低.提示p27蛋白表达降低与肝癌的发生和进展有着密切关系,并可能是导致肝癌细胞增殖凋亡失衡的因素之一.     

组织芯片检测肝肠钙粘连蛋白在肝癌组织中的表达及临床意义

目的研究肝肠钙粘连蛋白（LI—cadherin）在肝癌组织中的表达以及与肝癌的发生发展和临床病理特征之间的相关性。方法采用免疫组织化学sP法检测70例不同类型肝癌组织和5例正常肝组织标本中LI—cadherin的表达情况,并结合临床资料和相关病理参数进行统计分析。结果70例肝癌组织中LI—cadherin阳性表达39例,总阳性率为55．7％,5例正常肝组织中未见阳性表达;LI—cadherin的表达与年龄、性别、肿瘤分化程度以及有无远处转移无关（P〉0．05）,而与淋巴结转移及血管侵犯密切相关（P〈0．05）。结论LI—cadherin的表达与肝癌的发生、转移和浸润有关,LI—cadherin可能成为诊断肝癌发生、转移及临床分期的新生物学标志物,对于提高临床治疗效果具有指导意义。