Cloning of differentially expressed genes in human hepatocellular carcinoma and nontumor liver

INTRODUCTIONThe mechanism of hepatocellular carcinoma(HCC)is still unclear,although some genes have been found to play a role in the transformation of liver cells,and a variety of studies have described differences in gene expression which distinguished tumor from nontumor[1-6].The new genes,especially the functional genes directly related with tumor are still worth being found.The purpose of our study is to find the different genes between human liver tumor and normal tissues using suppression subtractive hybridization.     

应用基因芯片技术筛选肝细胞癌相关基因

目的:探讨基因表达谱芯片技术在筛查肝细胞癌(HCC)相关基因群表达中的作用。方法:TRIzol法抽提2例HCC及正常肝脏组织总RNA,分离纯化两种组织的mRNA;逆转录合成掺入生物素标记的cDNA合成探针,与基因芯片(涵盖了18400个转录本,代表了14 500个明晰的基因)杂交,扫描芯片荧光信号图像,计算机分析,比较2种组织基因表达谱差异。结果:2例HCC组织与正常肝脏组织相比,有2756条基因(19.01%)共同表达差异,其中共同上调基因1772条和共同下调基因984条,对2756条共同差异表达基因作了初步功能分类,这些基因与HCC的发病机制存在相关性。结论:基因表达谱芯片技术可以筛选出HCC表达异常的相关基因群,有助于认识肝癌的发病机制。     

Polymorphism of glutathione S-transferase mu 1 and theta 1 genes and hepatocellular carcinoma in southern Guangxi, China

AIM: Glutathione S-transferase mu 1 (GSTM1) and theta 1 (GSTT1) genes are involved in the metabolism of a wide range of carcinogens, but deletions of the genes are commonly found in the population. The present study was undertaken to evaluate the association between GSTM1 and GSTT1 gene polymorphisms and hepatocellular carcinoma (HCC) risk. METHODS: The genetic polymorphisms were studied at an aflatoxin highly contaminated region in Guangxi, China. Pdymerase chain reaction (PCR) technique was used to detect the presence or absence of the GSTM1 and GSTT1 genes in blood samples. The case group was composed of 181 patients of HCC identified by the pathologists and the control group was composed of 360 adults without any tumor. RESULTS: The frequencies of GSTM1 and GSTT1 null genotypes in the control were 47.8% and 42.7%, while those in the HCC group were 64.6% and 59.7%, respectively. The differences between HCC group and control group were very significant (P<0.01). GSTM1 and GSTT1 combined null genotypes in HCC group and control group were 38.2% and 18.5% respectively, and the difference was significant (P<0.05). CONCLUSION: The GSTM1 and GSTT1 null genotypes are associated with an increased risk of HCC in a special geographic environment. Combination of the two null genotypes in an individual is substantially increased twice the risk of HCC.     

正常肝组织与肝癌组织差异表达基因消减cDNA文库的构建

目的 构建人正常肝组织与肝癌组织差异表达基因的消减cDNA文库。方法 采用新近建立的抑制消减杂交技术,以癌旁正常肝组织及肝癌组织作为对比材料,分离肝癌组织中不表达或低表达基因的cDNA片段,将其与T载体进行T/A连接构建文库,将连接产物用电穿孔法转化大肠杆菌进行文库扩增后,随机挑取100个白色克隆进行酶切鉴定。结果 扩增消减cDNA文库获得4000余个白色阳性克隆,随机挑取的100个白色克隆进行酶切鉴定。结果 扩增消减cDNA文库获得4000余个白色阳性克隆,随机挑取的100个白色克隆经酶切后均有200-600bp的插入片段。结论 用SSH法及T/A克隆技术成功构建了正常肝组织与肝癌组织差异表达基因的消减cDNA文库,该文库的建立为进一步筛选、克隆肝癌组织中失活或低表达的新的抑癌基因奠定了基因。     

Human hepatitis B virus and hepatocellular carcinoma II. Experimental induction of hepatocellular carcinoma in tree shrews exposed to hepatitis B virus and aflatoxin B1

On the basis of the successful establishment of an animal model in tree shrews experimentally infected with human hepatitis B virus (HBV), a study on the hepatocarcinogenic effects of HBV and/or aflatoxin B1 (AFB1) was conducted. The results showed that the incidence of hepatocellular carcinoma (HCC) was significantly higher in the animals both infected with HBV and exposed to AFB1 (52.94%) than in those solely infected with HBV (11.11%) or exposed to AFB1 (12.50%). No HCC of precancerous lesions were found in the controls that were neither HBV-infected nor AFB1-exposed. Precancerous lesions, including liver cell dysplasia and enzyme-altered hyperplastic hepatocyte foci, were observed before the occurrence of HCC, and the frequency of their appearance correlated well with the incidence of HCC. HBV DNA and the protein it encodes were detected in the cancer cells and/or the surrounding hepatocytes. Integration of HBV DNA inot the host liver genome was found during hepatocarcinogenesis among the animals infected by HBV. These results suggest that exposure to HBV and AFB1 may play a synergistic role in the development of HCC, and support the viewpoint of an aetiological relationship between HBV and HCC.Abbreviations HCC hepatocellular carcinoma - HBV human hepatitis B virus - AFB1 aflatoxin B1 - GGT foci hyperplastic hepatocyte foci positive for -glutamyltranspeptidase - LCD liver cell dysplasia     

人肝细胞癌差异表达基因TCTP的克隆和分析

目的 筛选和鉴定在人肝细胞癌中差异表达的基因。方法 采用抑制性消减杂交技术建立人肝细胞癌eDNA消减文库，通过DNA测序分析、Northern印迹、cDNA末端快速扩增和RT-PCR等方法对其中一个克隆基因加以分析。结果 从所建eDNA消减文库中分离到一个插人子长度为479bp的克隆，该片段含有3’端Poly(A)结构，Northern杂交证明其表达水平在癌组织和肝癌细胞株均显著升高，同时经5’末端快速扩增获得一个长度为865bp的全长cDNA序列，具有一个编码172个氨基酸的完整开放阅读框，与GenBank数据库作同源性分析显示其为已报道的TCTP基因。RT-PCR分析表明TCTP基因在癌组织样本中的扩增水平高于癌旁非癌组织样本。结论‘I'CTP基因的差异表达可能在肝癌发生机制中扮演重要角色。     

生物信息学方法对人类和小鼠共同肝癌相关基因的筛选

目的:研究应用生物信息学技术,对基因芯片产生的大量数据进行系统地挖掘和分析,筛选出人类和小鼠不同种属间在肝癌发生发展过程中起关键作用的共同基因.方法:首先通过文献检索和收集,在GEO数据库中下载符合纳入标准的9套样本基因芯片数据,运用bioconductor和Rversion的2.10.1版本对已下载数据进行标准化处理,运用软件包affy中的RMA算法对affymetrix平台的原始数据进行背景校正、标准化和log2转换;其次使用excel的TTEST函数计算每一个基因的显著性,DAVID进行探针基因名称转换,建立基因名称与样本对应的表达数据表;再进行Meta分析计算人类及小鼠的共同差显基因,并通过DAVID中的KEGG库富集调控通路.结果:人类与小鼠在肝癌发生发展过程中的共同表达基因52个,其中5个为上调基因,4个为下调基因;富集出7条通路,其中甘氨酸、丝氨酸、苏氨酸代谢通路和轴突引导通路是文献已报道的与肝癌发生发展密切相关通路.结论:通过生物信息学可快速筛选出人类与小鼠在肝癌发生发展过程中的共同关键基因及与肝癌发生发展密切相关通路.     

Immune checkpoint blockade for the treatment of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers with a high recurrence rate. Currently, tyrosine kinase inhibitors (TKIs) are the first‐line treatment for cases refractory to conventional therapies. However, the acquisition of somatic mutations can result in TKI resistance. Clinical evidence suggests that acquired immunity contributes to the suppression of tumor recurrence, indicating the potential of induced antitumor immune reaction for the treatment of HCC. Recently, immune checkpoint inhibitors have become available for the treatment of malignancies. They are effective regardless of the response to prior therapies and a durable effect can be expected, which should be attributed to an adaptive immunity to HCC components. The results of phase I/II trials of nivolumab, an anti‐programmed cell death‐1 antibody, showed that 20% of patients showed objective response and that nivolumab was effective regardless of prior sorafenib treatment and viral status. Nivolumab received expedited Food and Drug Administration approval in 2017 for the treatment of advanced HCC after failure or intolerance to sorafenib. However, the majority of the patients remain refractory, likely due to the solid immune suppressive status, which involves many stromal cells, humoral mediators, and suppressive checkpoint molecules. Therefore, current clinical trials are focusing on how immunosuppressive conditions in HCC might be overcome using immune checkpoint inhibitors in combination with different types of immune checkpoint blockades, TKIs, and other conventional treatments. The development of immune checkpoint inhibitors is rapidly progressing and these inhibitors are likely to be key agents for HCC treatment in the near feature.     

膜联蛋白A2过表达在肝细胞癌发生发展中的作用

膜联蛋白A2与S100A10组装成异四聚体转位细胞膜从而激活相关信号通路或者Ser^25磷酸化以后进入细胞核参与DNA合成和细胞增殖,可调节血管形成、与肝细胞癌（HCC）形成与预后相关。本文综述了膜联蛋白A2的分子构成、功能及对HCC诊断和预后价值的研究进展。     

Role of hepatitis C virus in the development of hepatocellular carcinoma: transgenic approach to viral hepatocarcinogenesis

Overwhelming lines of epidemiologic evidence have indicated that chronic infection with hepatitis C virus (HCV) poses a major risk towards the development of hepatocellular carcinoma (HCC). It remains controversial whether HCV plays a direct role in the pathogenesis of HCV-associated HCC or whether it merely serves an indirect role. Using the transgenic mouse model established by us, it has become evident that the core protein of HCV confers oncogenic potential. The findings in our studies indicate that HCV is directly involved in hepatocarcinogenesis, albeit other factors, such as continued cell death and regeneration associated with chronic hepatitis, may also play a role. Taken together, our results indicate that there could be a mechanism for the development of HCC in persistent hepatitis virus infection that is distinct from the pathogenesis of other cancers, like colorectal cancer. Thus, although accumulation of a set of genetic aberrations may also be necessary for a multistage development of HCC, HCV core protein, to which an oncogenic potential is ascribed, may allow some stages to be skipped in hepatocarcinogenesis. The possibility that infection with HCV may be capable of inducing HCC in the absence of a complete set of genetic aberrations would help explain the unusually high incidence and multicentric nature of HCC developing in chronic hepatitis C.     

Comprehensive analysis of prognostic immune-related genes in the tumor microenvironment of hepatocellular carcinoma (HCC)

Growing evidence supports that the tumor microenvironment plays a key role in the development and progression of tumors. But immune microenvironment of hepatocellular carcinoma (HCC) has not yet been fully explored. In the present investigation, the clinical value and prognostic significance of immune-related genes in HCC were investigated.The immune and stromal scores of HCC were calculated through the application of Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data Algorithm based on the Cancer Genome Atlas database. Differentially expressed genes were identified using the “edgeR” package of the R software. Functional annotation and pathway enrichment were performed using “ggplots2” and “clusterProfiler” packages in R software. Protein-protein interaction network was constructed using STRING, and the hub genes were identified through the Cytoscape. Survival analysis was performed using Kaplan-Meier methods. Tumor Immune Estimation Resource algorithm was used to view the immune landscape of the microenvironment in HCC.Firstly, the immune and stromal scores of HCC were calculated and we found that the immune and stromal scores of HCC were closely related to the patients’ prognosis. Then the differentially expressed genes were identified respectively stratified by the median value of the immune and stromal scores, and the immune-related genes that related to the prognosis in HCC patients were further identified. Functional enrichment analysis and protein-protein interaction networks further showed that these genes mainly participated in immune-related biological process. In addition, dendritic cells were found to be the most abundant in the microenvironment of HCC through Tumor Immune Estimation Resource algorithm and were significantly associated with the patients’ prognosis. To robust the results, the immune-related genes were validated in an independent dataset from the Gene Expression Omnibus database.We arrived at a more comprehensive understanding of the microenvironment of HCC and extracted 7 immune-related genes that were significantly associated with the recurrence survival of HCC.     

同种异基因皮肤移植对昆明小鼠荷H22型肝癌细胞生长的影响

目的观察同种异基因皮肤移植对昆明小鼠肝细胞癌生长及相关免疫指标的影响。方法采用荷H22型肝癌昆明小鼠为动物模型并分为3组,在皮下接种小鼠H22肝癌细胞悬液后,接受C57／BL6小鼠皮肤移植物的昆明小鼠为实验组,而接受同种同基因移植和不移植组均作为对照组,比较肿瘤的生长情况;同时检测小鼠外周血淋巴细胞亚群和肿瘤组织内细胞的凋亡细胞,比较各组间差异。结果接受同种异基因皮肤移植的昆明小鼠,肿瘤的生长速度慢于其他2组;接受同种异基因皮肤移植的小鼠瘤组织内凋亡细胞数量高于对照组。结论同种异基因皮肤移植对小鼠H22肝癌细胞的生长具有抑制作用,这种作用可能通过诱导肝癌细胞凋亡而发挥作用。     

Cancer of the Liver Italian Program (CLIP) score for staging hepatocellular carcinoma

Aim:  To assess the prognostic ability of the Cancer of the Liver Italian Program (CLIP) score in patients with hepatocellular carcinoma after a longer follow up.
Methods:  The updated survival data were derived from an analysis that was performed on two joined sets of data. The first set was collected retrospectively in 1995 and was used to perform an exploratory prognostic factor analysis (the CLIP-03 study), that produced the CLIP score. The second set of data was collected prospectively for the CLIP-01 randomized clinical trial.
Results:  Out of 912 overall patients, analysis was performed on 650 patients whose records contained all informationregarding prognostic factors. The median survival of the whole group of patients was 17.7 months and the 5-year survival rate was 10.7%. The median survival of the patients was inversely proportional to the CLIP score: the higher the CLIP score the worse the survival.
Conclusions:  The CLIP score keeps good prognostic and discriminative abilities after a longer follow up and remains one the most useful prognostic system for hepatocellular carcinoma.     

Methylation of Ras association domain family protein 1, isoform A correlated with proliferation and drug resistance in hepatocellular carcinoma cell line SMMC-7721

Background: It has been proposed that aberrant DNA methylation can alter cancer's response to therapeutic agents. The purpose of the present study was to investigate the relationship between Ras association domain family protein 1, isoform A (RASSF1A) and drug resistance or growth of hepatocellular carcinoma cell line SMMC‐7721. Methods: Methylation status of RASSF1A from genomic gene of SMMC‐7721 was determined with methylation‐specific polymerase chain reaction and was further verified by demethylation treatment with 5‐aza‐2′‐deoxycytidine. Wild‐type RASSF1A cDNA was introduced into SMMC‐7721; chemosensitivity to cisplatin, mitomycin and fluorouracil were analyzed using 3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyltetrazolium bromide assay. Growth of cells was detected by colony formation assay and xenograft model. Results: RASSF1A was inactivated in SMMC‐7721 cell line through epigenetic hypermethylation, and reintroduction of RASSF1A was markedly able to reduce the resistance to some anticancer drugs and inhibit the growth of cancer cells either in vitro or in vivo. Conclusion: These results correlate methylation status of RASSF1A with biological profiles of SMMC‐7721 and thus, associate methylation with cancer therapy. RASSF1A may be useful to predict treatment response and help to develop novel treatment strategies for clinical treatment of hepatocellular carcinoma.