Dynamic alteration of telomerase expression and its diagnostic significance in liver or peripheral blood for hepatocellular carcinoma

AIM: To investigate the dynamic alteration of telomerase expression during development of hepatocellular carcinoma (HCC) and its diagnostic implications in liver tissues or peripheral blood mononuclear cells for HCC. METHODS: Dynamic expressions of liver telomerase during malignant transformation of hepatocytes were observed in Sprague-Dawly (SD) rats fed with 0.05% of 2-fluoenyacetamide (2-FAA). Total RNA and telomerase were extracted from rat or human liver tissues. The telomerase activities in livers and in circulating blood were detected by a telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA), and its diagnostic value was investigated in patients with benign or malignant liver diseases. RESULTS: The hepatoma model displayed the dynamic expression of hepatic telomerase during HCC development. The telomerase activities were consistent with liver total RNA levels (r = 0.83, P < 0.01) at the stages of degeneration, precancerosis, and cancerization of hepatocytes. In HCC patients, the telomerase levels in HCC tissues were significantly higher than in their adjacent non-cancerous tissues, but liver total RNA levels were lower in the former than in the latter. Although the circulating telomerase of HCC patients was abnormally expressed among patients with chronic liver diseases, the telomerase activity was a non-specific marker for HCC diagnosis, because the incidence was 15.7% in normal control, 25% in chronic hepatitis, 45.9% in liver cirrhosis, and 85.2% in HCC, respectively when absorbance value of telomerase activity was more than 0.2. If the value was over 0.6, the incidence was 60% in HCC group and 0% in any of the others (P < 0.01) except in two cases with liver cirrhosis. However, the combination of circulating telomerase with serum alpha-fetoprotein level could increase the positive rate and the accuracy (92.6%, 125 of 135) of HCC diagnosis. CONCLUSION: The overexpression of telomerase is associated with HCC development, and its abnormality in liver tissues or in peripheral blood could be a useful marker for diagnosis and prognosis of HCC.     

Expression of transforming growth factor-α and hepatitis B surface antigen in human hepatocellular carcinoma tissues and its significance

AIM:To evaluate the expression of transforming growthfactor-alpha (TGF-α) and hepatitis B surface antigen (HBsAg)in human hepatocellular carcinoma (HCC) tissues and itssignificance.METHODS:Seventy specimens of HCC tissues weredetected by immunohistochemical method.Five specimensof normal human liver tissues were used as control.RESULTS:The TGF-α positive expression rates in HCCand its surrounding tissues were 74.3%(52/70) and88.1%(52/59),respectively.TGF-α positive granules weremainly in the cytoplasm and fewer existed on thekaryotheca.The TGF-α positive expressing rate in welldifferentiated HCC was significantly higher than that inmoderately and poorly differentiated HCC (P<0.05).TheTGF-α positive expression also was observed in intrahepaticbile ducts (part of those were hyperplastic ducts).TheHBsAg positive expression rates in HCC and its surroundingtissues were 21.4%(15/70) and 79.7%(47/59),respectively.HBsAg positive granules were in the cytoplasm,inclusionand on the karyotheca.There was a prominent positivecorrelation between TGF-a and HBsAg expression in HCCsurrounding tissues (P<0.05,γ=0.34).TGF-α was usuallyexisted with HBsAg in regenerated and/or dysplastic livercells.In the five normal liver tissues,TGF-α and HBsAgwere not detectable in hepatocytes and bile ducts.CONCLUSION:Hepatitis B virus infection is closely relatedwith hepatocarcinogenesis.The overexpression of TGF-αin the liver seems to be associated with the regenerationof hepatocytes injured by HBsAg.The continued expressionof TGF-α might lead to dysplasia of liver cells anddevelopment of HCC.Furthermore,TGF-α might play arole in morphogenesis and regeneration of intrahepaticbile ducts.     

Expression of transforming growth factor-alpha and hepatitis B surface antigen in human hepatocellular carcinoma tissues and its significance

AIM:To evaluate the expression of transforming growthfactor-alpha (TGF-α) and hepatitis B surface antigen (HBsAg) in human hepatocellular carcinoma (HCC) tissues and its significance.METHODS:Seventy specimens of HCC tissues were detected by immunohistochemical method. Five specimens of normal human liver tissues were used as control.RESULTS: The TGF-o~ positive expression rates in HCC and its surrounding tissues were 74.3%(52/70) and 88.1%(52/59), respectively. TGF-α positive granules were mainly in the cytoplasm and fewer existed on the karyotheca. The TGF-α positive expressing rate in well differentiated HCC was significantly higher than that in moderately and poorly differentiated HCC (P&lt;0.05).The TGF-α positive expression also was observed in intrahepatic bile ducts (part of those were hyperplastic ducts).The HBsAg positive expression rates in HCC and its surrounding tissues were 21.4%(15/70) and 79.7%(47/59), respectively.HBsAg positive granules were in the cytoplasm, inclusion and on the karyotheca.There was a prominent positive correlation between TGF-α and HBsAg expression in HCC surrounding tissues (P&lt;0.05,γ=0.34). TGF-α was usually existed with HBsAg in regenerated and/or dysplastic liver cells.In the five normal liver tissues, TGF-α and HBsAg were not detectable in hepatocytes and bile ducts.CONCLUSION:Hepatitis B virus infection is dosely related with hepatocarcinogenesis.The overexpression of TGF-α in the liver seems to be associated with the regeneration of hepatocytes injured by HBsAg.The continued expression of TGF-α might lead to dysplasia of liver cells and development of HCC. Furthermore, TGF-α might play a role in morphogenesis and regeneration of intrahepatic bile ducts.     

Expression of gap junction genes connexin 32, connexin 43 and their proteins in hepatocellular carcinoma and normal liver tissues

AIM To investigate the significance and mechanism of cx32 mRNA, cx43 mRNA and their proteins in hepatocarcinogenesis.METHODS Sixty-one cases of HCC and 14 cases of normal liver tissues were detected by immunohistochemical and in situ hybridization (ISH) methods.RESULTS In HCC grades Ⅰ,Ⅱ,Ⅲ and normal liver tissues, the positive rates of Cx32 protein were 55.6%, 42.1%, 18.2% and 92.9%,respectively. The detection rates of Cx43 protein were 44.4%, 26.3%, 12.1% and 78.6%,respectively. There was significant difference in Cx32 and Cx43 protein between HCC and normal liver tissues (P＜0.01). ISH the positive rates of cx32 mRNA shown by ISH in HCC grades Ⅰ,Ⅱ,Ⅲ and normal liver tissues were 88.9%, 84.2%,87.9% and 92.9%, respectively. Those of cx43 mRNA were 77.8%, 78.6%, 78.8% and 85.7%,respectively. There was no statistical difference in the positive rates of cx32 mRNA and cx43 mRNA between HCC and normal liver tissue (P＞0.05).CONCLUSION The aberrant location of Cx32 and Cx43 proteins could be responsible for progression of hepatocarcinogenesis, and the defect of cx genes in post-translational processing might be the possible mechanism.     

Overexpression of HBxAg in hepatocellular carcinoma and its relationship with Fas／FasL system

AIM: To study the expression and serum level of HBxAg,Fas and FasL in tissues of HCC patients, and to assess the relationship between HBxAg and Fas/FasL system.METHODS: Tissues from 50 patients with HCC were tested for the expression of HBxAg, Fas and FasL by S-P immunohistochemistry. Serum levels of sFas/sFasL and HBsAg/HBeAg were measured by ELISA assay. HBV X gene was detected by PCR in serum and confirmed by automatic sequencing. Fifty cases of liver cirrhosis and 30 normal controls were involved in serum analysis.RESULTS: The expression of HBxAg, Fas and FasL in carcinoma tissues was 96 %, 84 % and 98 %, respectively.Staining of HBxAg, Fas and FasL was observed predominately in cytoplasms, no significant difference was found in intensity between HBxAg, Fas and FasL (P＞0.05). HBxAg, Fas and FasL might express in the same area of carcinoma tissues and this co-expression could be found in most patients with HCC. The mean levels of sFas in serum from HCC, cirrhosis and normal controls were 762.29±391.56 μg@ L-1 835.36±407.33 μg@L-1 and 238.27±135.29 μg@L-1. The mean levels of sFasL in serum from HCC, cirrhosis and normal controls were 156.36±9.61iμg@ L-1, 173.63±18.74 μg@L-1 and 121.96±7.83 μg@ L-1.Statistical analysis showed that both sFas and sFasL in HCC and cirrhosis patients were significantly higher than those in normal controls (P＜0.01). Serum HBV X gene was found in 32 % of HCC patients and ,46 % of cirrhotic patients.There was no significant relationship between serum level of sFas/sFasL and serum X gene detection (P＞0.05). Eight percent of HCC patients with negative HBsAg and HBeAg in serum might have X gene in serum and HBxAg expression in carcinoma tissues.CONCLUSION: Our data suggest that HBxAg and Fas/FasL system plays an important role in the development of human HCC. Expression of HBxAg can leads to expression of Fas/FasL system which and reverse apoptosis of hepatocellular carcinoma induced by FasL.     

Helicobacter infection in hepatocellular carcinoma tissue

AIM: To investigate whether Helicobacter species (Helicobacter spp.) could be detected in hepatocellular carcinoma (HCC) tissue. METHODS: Liver samples from 28 patients with hepatocellular carcinoma (HCC) diagnosed by histopa-thology were studied. Twenty-two patients with other liver diseases (5 with liver trauma, 7 with cavernous liver hemangioma, 6 with liver cyst and 4 with hepatolithiasis), 25 patients with gastric cancer, 15 with colonic cancer and 15 with myoma of uterus served as controls. Two piceces of biopsy were obtained from each patient. One was cultured for Helicobacter spp. and extraction of DNA, the other was prepared for scanning electron microscopy (SEM) and in situ hybridization. The samples were cultured on Columbia agar plates with microaerobic techniques. Helicobacter spp. in biopsy from the studied subjects was detected by polymerase chain reaction (PCR) with Helicobacter spp. 16S rRNA primers. Amplified products were identified by Southern hybridization and sequenced further. Besides, other genes (vacA, cagA) specific for Helicobacter pylori (H pylori) were also detected by PCR. Helicobacter spp. in biopsies was observed by SEM. Transmission electron microscopy (TEM) was performed to identify the cultured positive Helicobacter spp. The presence of Helicobacter spp. was detected by in situ hybridization to confirm the type of Helicobacter. RESULTS: The positive rate of Helicobacter cultured in HCC and gastric cancer tissue was 10.7% (3/28) and 24%(6/25), respectively. Helicobacter microorganisms were identified further by typical appearance on Gram staining, positive urease test and characteristic colony morphology on TEM. The bacterium was observed in adjacent hepatocytes of the two HCC samples by SEM. The number of cocci was greater than that of bacilli. The bacterium was also found in four gastric cancer samples. PCR showed that the positive rate of HCC and gastric cancer samples was 60.7% and 72% respectively, while the controls were negative (P<0.01). The PCR-amplified products were identified by Southern hybridization and sequenced. The homology to 16S rRNA of H pylori was 97.80%. The samples were verified by in situ hybridization for Helicobacter spp. 16S rRNA mRNA and proved to be H pylori positive. There was no statistical significance between HCC and gastric cancer (P>0.05), but the positive rate of HCC and controls had statistical significance (P<0.01). Only 3 HCC samples and 2 gastric cancer samples of the cagA genes were detected. None of the samples reacted with primers for vacA in the two groups. As for the genotype of H pylori, typeⅡhad preference over typeⅠ. CONCLUSION: Helicobacter infection exists in liver tissues of HCC patients. Helicobacter spp. infection is related with HCC, which needs further research.     

内皮细胞特异性分子1在原发性肝癌中的表达及其意义

Objective To study the expression and significance of endothelial cell specific molecule-1(ESM-1) in hepatocellular carcinolna(HCC)tissue and serum from HCC patients.Methods The ESM-1 expression was evaluated by immunohistochemistry staining and quantitative real-time PCR in 30 tumor tissues from HCC patients,15 surrounding non-cancerous hepatic parenchyma,and 15 normal controls.Serum levels of ESM-1 were also measured in the HCC patients and healthy controls by Enzyme-linked imrnunosorbent assay(ELISA).Results ESM-1 was expressed in endothelium of 24 HCC tissues and 6 pericarcinomatous tissues.however,it was not expressed in normal liver tissues.The mRNA level of ESM-1was 0.064±0.018,0.383±0.103,0.528±0.148 in the corresponding tissues,respectively.The mRNA level of ESM-1 in tissues was correlated with the TNM phase of HCC patients.tumor vascular invasion as well as metastasis.The serum ESM-1 was(12.643±2.280)ng/ml and(4.660±1.172)ng/ml in hepatic cell carcinoma and normal controls,respectively.Conclusion The expression of ESM-1 is significantly upregulated in HCC,suggesting that ESM-1 may play an important role in the pathoigenesis of HCC.     

转化生长因子β1诊断肝癌和肝癌监测转移的临床价值

目的观察肝癌发生过程中TGFβ1及基因动态表达的临床价值。方法以2-乙酰氨基芴（2-FFA）喂饲雄性SD鼠诱发肝细胞癌变，以病理学方法、巢式RT-PCR、免疫组织化学法和ELISA，观察肝细胞形态学、TGFβ1 mRNA、TGFβ1胞内定位、肝组织及外周血TGFβ1的动态变化与临床价值。结果SD鼠在喂饲2-FAA后，肝细胞出现颗粒样变性、不典型增生到肝癌形成。肝和血TGFβ1：正常组分别为（1．97±0．28）ng／mg和（5．98±1．76）ng／ml，变性组分别为（2．56±0、31）ng／mg和（9、41±0．37）ng／ml，癌前组分别为（3．88±0．11）ng／mg和（12．89±0、69）ng／ml，癌变组分别为（6、74±1．41）ng／mg和（16．74±2．19）ng／ml。癌变组明显高于对照组、肝细胞变性组和癌前病变组。癌变过程中TGFβ1阳性表达呈胞内分布，肝和外周血中TGFβ1呈显著正相关。肝癌患者血浆TGFβ1诊断肝癌的阳性率为89、5％，在血AFP〈400μg／L肝癌患者中，TGFβ1阳性率为93．3％，与AFP联合检查诊断肝癌阳性率达94．7％。肝癌组织TGFβ1 mRNA呈强阳性表达，伴有肝外转移患者外周血TGFβ1 mRNA检出率为100．0％。结论TGFβ1参与肝细胞癌变过程，TGFβ1及TGFβ1 mRNA过表达有助于肝癌早期诊断及预后判断。     

Expression and significance of tumor-related genes in HCC

AIM: To investigate the expression and clinical significance of DEK, cyclin D1, insulin-like growth factor Ⅱ (IGF-Ⅱ), glypican 3 (GPC3), ribosomal phosphoprotein 0 (rpPO) mRNA in hepatocellular carcinoma (HCC) and its paraneoplastic tissues. METHODS: The expression of mRNAs of DEK, cyclin D1, IGF-Ⅱ, GPC3 and rpP0 mRNA was detected in HCC and its paraneoplastic tissues by multiplex RT-PCR. RESULTS: By the simplex RT-PCR, the overexpression of mRNAs of DEK, cyclin D1, IGF-Ⅱ, GPC3, rpP0 mRNA in HCC and its paraneoplastic tissues was 78.1%, 87.5%, 87.5%, 75.0%, 81.3% and 15.6%, 40.6%, 37.5%, 21.9%, 31.3% respectively (P<0.05). By the multiplex RT-PCR, at least one of the mRNAs was detected in all HCC samples and in 75.0% of paraneoplastic samples (P>0.05). However, all these five mRNAs were found in 68.8% of HCC samples, but only in 9.4% of paraneoplastic tissues (P<0.05). The positive expression of mRNAs of DEK, cyclin D1, IGF-Ⅱ, GPC3, rpP0 in well- and poorly-differentiated HCC was 89.0%, 66.7%, 66.7%, 66.7%, 77.8% and 73.9%, 95.7%, 95.7%, 95.7%, 82.6%, respectively (P>0.05). The expression of these genes in HCCs with α-feto protein (AFP) negative and positive was 90.0%, 80.0%, 90.0%, 90.0%, 90.0% and 72.7%, 86.3%, 77.3%, 90.9%, 68.2% respectively (P>0.05). CONCLUSION: The expression of DEK, cyclin D1, IGF-Ⅱ, GPC3, rpP0 mRNA in HCC is much higher in HCC than in its paraneoplastic tissues. Multiplex RT-PCR assay is an effective, sensitive, accurate, and cost-effective diagnostic method of HCC.     

原发性肝癌患者外周血中甲胎蛋白mRNA的意义

目的由于ＰＣＲ技术的应用,血循环中癌细胞的检测近有很大进展．本文用ＲＴＰＣＲ检测肝癌（ＨＣＣ）和其他慢性肝病患者外周血中ＡＦＰｍＲＮＡ,藉以反映ＨＣＣ细胞的存在,并与其他血清标记物比较．方法ＨＣＣ患者２２例,肝硬变和慢性乙型肝炎患者各１０例,健康成人受试者（对照）５例．取患者和对照的外周血,分离单核细胞,提取总ＲＮＡ并作电泳鉴定,用合成的两对引物进行巢式ＲＴＰＣＲ扩增ＡＦＰｍＲＮＡ,同时分析血清ＡＦＰ和乙肝标记物．结果ＡＦＰｍＲＮＡ在１３例ＨＣＣ（５９１％）,２例肝硬变（２００％）患者外周血中测到,其余标本均为阴性．ＡＦＰｍＲＮＡ阳性的１３例患者肿瘤均大于５ｃｍ,为晚期患者．在该１３例患者中仅有６例（４６１％）在血清中测到ＡＦＰ,但有１２例（９２３％）ＨＢｓＡｇ,抗ＨＢｅ,抗ＨＢｃ全阳性,而ＡＦＰｍＲＮＡ阴性的５例该３标记物全阴性．结论ＲＴＰＣＲ扩增ＡＦＰｍＲＮＡ是检测ＨＣＣ和肝硬变患者循环肝癌细胞的敏感方法．患者外周血中的ＡＦＰｍＲＮＡ有可能作为肿瘤转移和复发的标记对ＨＣＣ诊断、随访观察和疗效评定有较大临床意义．     

缺氧诱导因子1α在肝癌中的表达及意义

目的检测肝癌组织及培养的肝癌细胞株HepG2细胞中缺氧诱导因子1α（HIF1α）的表达及意义.方法用免疫组织化学、Western blot和逆转录聚合酶链反应（RT-PCR）法检测肝癌和正常肝组织及HepG2细胞中HIF1α蛋白质和mRNA的表达,并分析其与肝癌病理特点的关系.结果免疫组织化学显示HIF1α在肝癌组织中表达明显,阳性率达76.4%,显著高于正常肝组织;其表达与肿瘤的分化程度、有无癌栓有关（P＜0.05）,而与门静脉癌栓、乙型肝炎表面抗原及预后无关（P值均＞0.05）;Western blot 和RT-PCR检测结果与免疫组织化学结果一致.HIF1 α在HepG2细胞中表达的阳性率为93.6%.缺氧或加二氯化钴（150μmol/L）作用2 h后HIF1 α蛋白质及mRNA表达增加.结论HIF1α蛋白质在肝癌中表达明显,在癌组织中表达主要受氧的调节,与肿瘤分化程度及有无转移有关,而与有无门静脉癌栓、乙型肝炎表面抗原表达及预后无关.     

Expression of IGF-II in early experimental hepatocellular carcinomas and its significance in early diagnosis

AIM: To investigate the serum level and expression of insulin growth factor II (IGF-II) in liver tissues of rats with early experimental hepatocellular carcinomas (HCC) and its significance in early diagnosis. METHODS: Early experimental hepatocellular carcinomas were induced by diethylnitrosamine (DENA) in 180 male SD rats. Another 20 male SD rats served as control. The IGF-II serum level was measured by ELISA. Immunohistochemistry and electron microscopic immunohistochemistry were used to observe the expression of IGF-II in normal and tumor liver tissues and its ultrastructural location in malignant hepatocytes. The expressions of IGF-II in human hepatoma cell lines HepG2, SMMC7721 and human embryonic liver cell line L-02 were measured by immunocytochemistry. IGF-II mRNA level was studied by in situ hybridization. RESULTS: IGF-II was expressed in the cytoplasm of both sinusoidal cells in paracancerous cirrhotic liver tissue and malignant hepatocytes in early experimental HCC tissues. Gold particles were seen on the rough endoplasmic reticulum and the mitochondrion in malignant hepatocytes. IGF-II was expressed in the human hepatoma cell lines. The mRNA level of IGF-II was higher in rat liver tumor tissue than in normal rat liver tissue. The serum IGF-II level of the early experimental HCC group was 34.67+/-10.53 ng.ml(-1) and that of the control group was 11.75+/-5.84 ng.ml(-1). The rank sum test was used for statistical analysis. There was a significant difference between the two groups (P<0.01). CONCLUSION: During the induction of early experimental HCC by DENA, IGF-II may promote hepatocytic proliferation via a paracrine mechanism in the pre-cancerous stage. When hepatocytes are transformed into malignant cells, they may secrete IGF-II and promote malignant cell proliferation by an autocrine mechanism. IGF-II may be a possible biological marker in the early diagnosis of HCC.     

Alteration of oncogenic IGF-II gene methylation status associates with hepatocyte malignant transformation

BackgroundOncogenic insulin-like growth factor-II (IGF-II) is overexpressed in hepatocellular carcinoma (HCC). The present study aimed to analyze the dynamic alteration of IGF-II CpG site methylation status and its molecular mechanism in HCC progression.MethodsIGF-II alterations were observed in rat hepatocarcinogenesis models induced by 2-acetylaminofluorene. Liver IGF-II expression was compared by immunohistochemistry or tissue IGF-II specific concentration (nmol/mg protein). Status of human IGF-II promoter 3 (P3) or rat IGF-II P2 CpG site methylation was amplified by methylation-specific polymerase chain reaction (MSP). Serum IGF-II levels were quantitatively detected by an enzyme-linked immunosorbent assay.ResultsThe levels of hepatic IGF-II expression were significantly elevated in the HCC group (P < 0.001). The unmethylation rate of IGF-II P3 CpG sites was 100% in the HCC-, 52.5% in the paracancerous-, and none (0%) in the distal noncancerous-tissues. Abnormal IGF-II expression was related to differentiation degree, tumor invasion, and positive HBV-DNA (all P < 0.001), with a negative correlation between P3 methylation degree and IGF-II expression. There was a positive correlation between liver IGF-II specific concentration and circulating IGF-II level (r = 0.97, P < 0.001). Significantly negative correlation was found between IGF-II P2 CpG site methylation and circulating IGF-II (r_s = ?0.89, P < 0.001) or liver IGF-II level (r_s = ?0.84, P < 0.001).ConclusionsThe increase of serum IGF-II and the alteration of oncogenic gene IGF-II methylation may be biomarkers for HCC diagnosis and DNA methylation may be the therapeutic target of HCC.