基于RNA-Seq技术的跨种属跨病因肝癌差异表达基因的筛选

目的:运用全转录组测序(RNA sequencing,RNA-Seq)技术和跨种属、跨病因筛选策略,定位出影响肝癌发生发展的关键因子.方法:运用RNA-Seq技术,对人肝癌、癌旁和正常肝组织,以及乙型肝炎病毒(hepatitis B virus,HBV)和黄曲霉毒素B1(aflatoxins,AFB1)诱发的树鼩肝癌、癌旁和正常肝组织标本进行全转录组测序、基因表达水平分析和比较;分别筛选出人肝癌差异表达分子、HBV和AFB1致癌的树鼩肝癌差异表达因子,最后定位出跨人和树鼩两个物种、跨HBV和AFB1两种致癌因素的肝癌共同差异表达分子.结果:各标本总RNA提取质量合格,RNASeq测序数据质量合格,测序数据与参考基因对比匹配率合格,样品间基因表达相关性合格.人肝癌差异表达基因有68个,其中上调基因14个,下调基因54个;HBV诱发的树鼩肝癌差异表达基因有314个,AFB1诱发的树鼩肝癌差异表达基因有20个,HBV和AFB1诱发的树鼩肝癌共同差异表达基因11个,均为下调基因.人肝癌和树鼩肝癌共同差异表达基因为2个,分别是载脂蛋白F(apolipoprotein F,APOF)和人胰岛素样生长因子酸不稳定亚基(insulin-like growth factor binding protein,acid labile subunit,IGFALS),其mRNA表达水平在肝癌中均下调.结论:运用RNA-Seq技术,跨种属、跨病因筛选肝癌关键基因的研究策略有可能推动肝癌关键基因的定位;APOF和IGFALS有可能是影响肝癌发生发展的重要分子.     

PeroxiredoxinⅡ基因在肝癌组织中的表达及意义

目的观察peroxiredoxinⅡ（PrxⅡ）mRNA和蛋白在黄曲霉毒素B1（AFB1）诱发树鼩HCC形成过程中的表达变化,并在人肝癌癌旁组织和正常肝组织中进行验证,以探讨PrxⅡ在肝癌形成过程中的作用。方法应用RT-PCR和Western blot方法,检测6例AFB1诱发的树鼩肝癌、癌旁组织和这些动物肝癌发生前的活检肝组织,以及6例对照组动物相应时期的肝活检组织PrxⅡ在mRNA水平和蛋白水平的表达情况,并在18例人肝癌及其相应癌旁组织以及17名正常人肝组织进行验证。结果PrxⅡ在树鼩肝癌组织中的mRNA和蛋白质表达水平均明显高于相应的癌旁和癌前组织,也高于对照组同期活检肝组织（P〈0．05）;人肝癌组织中PrxⅡ的mRNA和蛋白表达改变和树鼩相符,即肝癌中的表达高于癌旁组织和正常肝组织（P〈0．05）。结论PrxⅡ基因可能与肝癌的发生发展有关,并有可能成为防治肝癌的分子靶标。     

用树鼩cDNA芯片研究树鼩肝癌中参与信号传导的部分基因变化情况

目的利用树鼩cDNA芯片研究黄曲霉毒素B1（AFBl）和（或）乙型肝炎病毒（HBV）引起的树鼩肝细胞癌（HCC）发生过程中参与信号传导的部分基因表达变化情况，从而进一步探讨HCC发生的分子机制。方法实验树鼩分3组：A组（AFB1组）、B组（AFB1＋HBV组）、C组（正常对照组）。所有动物在实验过程中定期接受剖腹手术取肝组织检查，至肝癌形成时处死动物取肝癌和癌旁组织。用树鼩cDNA芯片检测实验第30、60、90周肝活检组织、肝癌组织及其癌旁组织中各基因的表达情况，并用实时逆转录聚合酶链反应（Real time RT-PCR）法验证cDNA芯片结果。结果在A组和B组从癌前到癌变过程中胰岛素样生长因子-Ⅱ（IGF-Ⅱ）、C-rel、核因子-κB2（NF—κB2）均显示有差异表达，同时bcl-2、细胞周期素A（cyclin A）、睫状神经营养因子（CNTF）仅在B组显示有差异表达。而C组这几个基因无差异表达。实验组与对照组比较，在诱癌的早、中期（30、60周）均出现CNTF及cyclin A的差异表达。realtime RT PCR结果与cDNA芯片检测结果基本一致。A组IGF-Ⅱ、C—rel基因及B组IGF-Ⅱ基因，在肝癌组织表达水平明显低于癌旁及实验30、60周组织，而癌旁与实验30、60周相比较则无明显差异；B组CNTF基因在癌旁、肝癌及60周之间比较无明显改变，但均明显高于30周；A组CNTF基因表达水平在癌旁、肝癌组织高于癌前组织，但差异无统计学意义；C组这3个基因不同时期比较亦无明显差异。结论树目自部分信号传导通路相关cDNA芯片应用于检测树目目HCC发生过程中信号传导通路中各基因表达的变化，对进一步了解树鼠甸HCC发生的机制有重要实用价值。IGF-Ⅱ、NF-κB2、C-rel、Bcl-2、cyclin A及CNTF这几个基因与树HCC的发生发展密切相关。     

表皮型脂肪酸结合蛋白在跨种属肝癌组织中的表达

目的 应用跨种属肿瘤关键基因筛选策略,从已报道的大量肝癌相关基因中筛选、定位出影响肝癌发生和发展的关键分子.方法 结合本课题组的前期实验数据及国内外文献数据,建立肝癌差异表达分子的跨种属数据集;对首批筛选出的候选分子表皮型脂肪酸结合蛋白(E-FABP),应用RT-PCR和Western blot技术,分别验证其在人、树鼩和大鼠的肝癌、癌旁及正常肝组织中mRNA和蛋白水平的差异表达情况.实验数据以均数±标准差((x)±s)表示,采用单因素方差分析. 结果 初步建成跨种属肝癌差异表达数据集,RT-PCR检测结果显示E-FABP mRNA在人肝癌及其癌旁组织和正常肝组织中的相对表达量分别为0.87±0.14、0.64±0.12和0.67±0.07;在树鼩肝癌及其癌旁组织和正常肝组织中的相对表达量分别为0.87±0.25、0.73±0.19和0.68±0.19;在大鼠肝癌及其癌旁组织和正常肝组织中的相对表达量分别为0.97±0.22、0.78±0.16和0.80±0.13.人、树鼩和大鼠肝癌组织中的E-FABP mRNA表达水平均显著高于癌旁和正常肝组织,F值分别为20.910、3.807、4.482,P值均＜0.05,差异有统计学意义.Wesern blot检测结果显示E-FABP在人、树鼩和大鼠肝癌组织中的蛋白表达水平均较癌旁和正常肝组织显著上调.结论 跨种属肿瘤关键基因筛选策略可能有助于发现肝癌关键基因,E-FABP有可能是影响肝癌发生和发展的重要分子之一.     

STAT3基因在原发性肝癌组织中的表达及意义

目的探讨STAT3基因在肝癌发生发展中的作用及意义。方法应用RT-PCR检测STAT3基因及相关基因c-myc、p53、survivin、VEGF的mRNA水平的表达,应用Western Blot法及免疫组化法检测STAT3基因的蛋白水平及定位表达。结果肝癌组织及癌旁组织中STAT3基因mRNA的表达均明显高于正常肝组织(P<0.05),肝癌及癌旁组织中c-myc、survivin、VEGF基因mRNA的表达上调,p53基因mRNA的表达下调,肝癌组织及癌旁组织中STAT3基因蛋白水平的表达均高于正常肝组织(P<0.05)。结论STAT3基因的持续激活在肝癌的发生发展中起重要作用,可作为早期诊断的指标及治疗的新靶点。     

乙肝相关性肝癌及癌旁组织p73基因表达及突变的研究

目的　探讨p73基因在人乙肝相关性肝癌组织中的表达及其意义。方法　采用RT PCR方法检测p73基因mRNA在癌组织、癌旁组织及正常肝组织中的表达差异 ;采用SSCP检测方法比较分析p73基因在肝癌发生中的变异情况。结果　癌组织中p73基因mRNA的转录水平明显高于癌旁组织及正常组织 (P <0 0 5 ) ;然而 ,肝癌组织、癌旁组织及正常肝组织中p73基因PCR扩增片段的多态性是一致的 ,电泳未见异常条带 (P >0 0 5 )。结论　新近发现的肿瘤抑制基因p73基因在乙肝相关性肝癌组织中末见明显的基因突变 ,相反表达水平明显增高 ,可见 ,p73基因以一种高表达形式参与乙肝相关性肝癌的发生     

C-myc基因和P16基因在肝细胞癌中的表达及其临床意义

目的探讨C-myc基因和P16基因表达在肝细胞癌(HCC)中的临床意义。方法采用免疫组化检测技术检测不同肝癌组织、癌旁组织及正常肝组织中的C-myc基因和P16基因表达产物。结果C-myc基因表达产物在肝癌组织中的表达明显高于癌旁组织和正常肝组织(P<0.05)。P16基因表达产物则在正常肝组织中和癌旁组织中表达明显高于肝癌组织(P<0.05)。C-myc基因的表达与HCC的发病年龄、性别、肿瘤大小、UICC分期无关(P>0.05),而与HCC的转移有关(P<0.05);P16基因的表达与HCC的发病年龄、性别、肿瘤大小无关(P>0.05),而与HCC的转移和UICC分期有关(P<0.05)。结论C-myc基因表达增强与P16基因表达障碍能促进HCC的发生发展,并可用来判断预后。     

肝癌相关差异基因的生物信息学分析

目的采用生物信息学方法对肝癌差异表达基因进行基因功能和信号通路分析,为临床筛选肝癌发生、发展的相关分子标志物及药物靶点提供理论基础。方法从公共数据库(GEO)中获取18例肝癌组织和18例癌旁组织的基因表达阵列数据进行生物信息学分析,筛选差异表达基因。利用DAVID和STRING数据库对差异表达基因进行基因功能、信号通路和蛋白相互作用分析。结果通过对两组数据的差异表达基因筛选,共获得1 108个差异表达基因,其中上调基因605个、下调基因503个。差异表达基因主要涉及细胞周期、DNA复制、癌通路、p53信号通路、黏附、补体途径、三大营养物质代谢及性激素代谢等。通过STRING数据库分析列出位于前20位蛋白互作网络的中心节点蛋白。结论本研究采用生物信息学方法对肝癌基因芯片数据进行挖掘,从基因水平探讨肝癌发生发展的物质基础、分子功能和生物学过程,为肝癌发生的机制研究、肿瘤标志物的筛选及药物靶点选择提供参考。     

肝癌中细胞凋亡水平和p73基因转录表达的研究

目的　通过观察肝癌组织 ,对应癌旁组织和远癌肝组织中细胞凋亡及p73基因转录表达水平的变化 ,探讨细胞凋亡及p73基因转录表达水平与肝癌发生、发展的关系。方法　采用TUNEL及RT_PCR技术检测 3 2例乙肝相关肝癌癌组织、癌旁组织 ,以及良性肝脏疾病和对应正常肝组织细胞凋亡和p73mRNA的表达水平 ,并结合临床病理学资料进行统计学分析。结果　①肝癌癌组织中的凋亡指数明显高于癌旁组织和远癌组织 (P <0 0 1) ,细胞凋亡水平与肝癌组织学类型和P_TNM分期无明显关系 (P >0 0 5) ;②肝癌组织p73mRNA表达水平和表达率明显高于癌旁肺组织、远癌组织和肝良性病变组织 (P <0 0 1) ;③组织中凋亡指数的升高和p73基因转录表达水平的增加呈显著的正相关 (P <0 0 1)。结论　肝癌组织中存在细胞调亡水平的升高和p73基因的过度转录表达     

应用基因芯片研究肝细胞癌组织差异基因表达谱

目的：应用基因芯片技术研究原发性肝细胞癌组织中的差异基因表达谱改变，以寻找肝细胞癌相关基因。方法：抽提正常肝组织和肝癌组织中的mRNA来制备探针，经杂交、洗涤后，通过计算机扫描分析正常肝组织和肝癌组织基因表达谱的差异情况。结果：在10000个候选基因中，筛选出102条差异表达基因，表达上调的有42条，表达下调的有60条。未知基因12条。结论：基于DNA微阵矩技术的肿瘤基因表达谱分析能够高通量筛选与肝癌发生发展相关的基因。     

Differential expression of genes during aflatoxin B(1)-induced hepatocarcinogenesis in tree shrews

AIM:Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B1 (AFB1),to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism.METHODS:Tree shrews ( Tupaia belangeri chinensis)were treated with or without AFB1 for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding noncancerous liver tissues, 2 biopsied tissues at the early stage(30^th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0^th week) of the experiment, and another i mixed sample of two liver tissues from the untreated control animals biopsied at the 90^th week of the experiment. The samples were then tested with the method of Atlas^TM cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared.RESULTS:The profiles of differently expressed genes were quite different in different ways of comparison.At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up-or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as “important genes” because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC,genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC.CONCLUSION: A considerable number of genes could change their expressing levels both in HCC and in HCC-surrounding non-cancerous liver tissues. A few modular genes were up-regulated only in HCC but not in surrounding liver tissues, while some apoptosis-related genes were down-regulated in HCC and up-regulated in surrounding liver tissues. To compare gene-expressing levels among the liver tissues taken at different time points during hepatocarcinogenesis may be helpful to locate the responsible gene (s) and understand the mechanism for AFB1 induced liver cancer.     

Dynamic expression of apoptosis-related genes during development of laboratory hepatocellular carcinoma and its relation to apoptosis

AIM:To explore the expression of p53,bcl-2,bax,survivin and the cell apoptosis during the development of tree shrew hepatocellular carcinoma(HCC),the relationship between expression of these genes,its impact on HCC development,and its relation to cell apoptosis.METHONS:Tree shrew HCC was induced with aflatoxin B1(AFB1),and regular biopsy of liver tissues was carried out and the biopsy tissues were collected during cancer inducement.Liver biopsy tisue and HCC tissue were collected from 35pre-cancerous experimental animats at wk 30 and 60 and at the 30th_,60th_,and 90th-wk,Liver biopsy tissues were collected from 13 blank control animals at wk 30,60,and 90.Expression of p53,bcl-2,bax,and survivin at each stage was examined by immunohistochemistry method.Apoptotic cells were detected in situ by the terminal deoxynucleotidyl transferase-mediated nick end labeling(tunel)technique.RESULTS:The apoptosis rate of normal hepatic cells was extremely low,whereas it increased during the formation of HCC.Expression of the apoptosis-related genes p53,bd-2,bax,and suvivin during the formation of HCC presented an increasing tendency.Expression of p53 did not noticeably relate to that of bcl-2,bax,and survivin,whereas expression of bcl-2 and bax was closely related.In HCC,p53 did not present a distinct relation to cell apoptosis,whereas its high level expression was probably related to liver cell proliferation.Survivin negetively correlated apoptosis index,and its overexpression could inhibit cell apoptosis.CONCLUSION:Apoptosis-related genes p53,bcl-2,bax,and survivin are all related to the occurrence of HCC.The anti-apoptosis effect of bcl-2 is influenced by bax,and ratio bcl/bax reflects more correctly the extent of cell apoptosis.     

树Qu实验性肝癌发生过程p53基因的变化

目的探讨由人乙型肝炎病毒(HBV)和黄曲霉毒素B1(AFB1)诱发的树鼩肝细胞癌变过程,p53基因的表达及变化.方法将树鼩分为四组A组HBV+AFB1,B组只感染HBV;C组只摄入AFB1;D组作空白对照.定期肝活检,用免疫组织化学、分子生物学等技术对实验树鼩肝及肿瘤组织进行检测.结果 (1)接受HBV及AFB1双因素的A组,肝细胞癌(HCC)发生率(66.7%)明显高于只接受HBV的B组或AFB1的C组(30%),而且HCC的平均发生时间也明显早于C组,(120.0±16.6)周与(153.3±5.8)周,t=3.336,P＜0.01.(2)在第75周前各组动物肝均未检出突变的p53蛋白.(3)105周时,A组p53蛋白表达率为78.6%,B组为60%,C组为71.4%,D组为10%(x2≥5.03,P＜0.05).在A、C组检出p53基因异常带.(4)树鼩肝癌p53基因突变点分别位于2 7 5、7 8及1 3密码子;其野生型p 5 3基因的核苷酸及氨基酸序列与人的p 5 3基因的核苷酸及氨基酸序列的同源性分别为91.7%、93.4%.结论再次证实HBV和AFB1有协同致肝癌作用;突变的p53蛋白出现于肝细胞发生癌变之前,p 5 3基因的突变促进了肝癌的发生和演进.HBV可能协同AFB1致p 5 3基因突变.     

Expression of IGF- II, p53, p21 and HBxAg in precancerous events of hepatocarcinogenesis induced by AFB1 and/or HBV in tree shrews

INTRODUCTION In order to study the relationship between oncogene expression and HCC generation, we observed the precancerous hepatic GGT foci, IGF-Ⅱ, p53 and p21 expression during hepatocarcinogenesis of tree shrew induced by hepatitis B virus (HBV) and/or aflatoxin B1 (AFB1).     

Dynamic expression of apoptosis-related genes during development of laboratory hepatocellular carcinoma and its relation to apoptosis

AIM: To explore the expression of p53, bcl-2, bax, survivin and the cell apoptosis during the development of tree shrew hepatocellular carcinoma (HCC), the relationship between expression of these genes, its impact on HCC development, and its relation to cell apoptosis. METHODS: Tree shrew HCC was induced with aflatoxin B1 (AFB1), and regular biopsy of liver tissues was carried out and the biopsy tissues were collected during cancer inducement. Liver biopsy tissue and HCC tissue were collected from 35 pre-cancerous experimental animals at wk 30 and 60 and at the 30th-, 60th-, and 90th-wk. Liver biopsy tissues were collected from 13 blank control animals at wk 30, 60, and 90. Expression of p53, bcl-2, bax, and survivin at each stage was examined by immunohistochemistry method. Apoptotic cells were detected in situ by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique. RESULTS: The apoptosis rate of normal hepatic cells was extremely low, whereas it increased during the formation of HCC. Expression of the apoptosis-related genes p53, bd-2, bax, and survivin during the formation of HCC presented an increasing tendency. Expression of p53 did not noticeably relate to that of bcl-2, bax, and survivin, whereas expression of bcl-2 and bax was closely related. In HCC, p53 did not present a distinct relation to cell apoptosis, whereas its high level expression was probably related to liver cell proliferation. Survivin negatively correlated apoptosis index, and its overexpression could inhibit cell apoptosis. CONCLUSION: Apoptosis-related genes p53, bcl-2, bax, and survivin are all related to the occurrence of HCC. The anti-apoptosis effect of bcl-2 is influenced by bax, and ratio bcl/bax reflects more correctly the extent of cell apoptosis.     

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.