阵列文库及阵列文库消减杂交用于肝癌相关基因筛选、表达和转录调控的初步研究
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摘要
原发性肝癌的研究在我国基础和临床医学研究中占有重要的地位，肝癌相关基因的克隆、鉴定、表达检测和转录调控对于从基因水平认识肝细胞的生长、分化、再生和恶变的分子机理、阐明其信号转导过程和途径，不仅具有重要的研究意义，而且对于肝癌的早期基因诊断和治疗具有重大的临床应用价值和经济利用价值。本文建立了基于DNA阵列的阵列文库消减杂交技术(Arrayed Library Subtraction Hybridization,ALSH)。以此技术结合基因芯片技术用于cDNA文库的大规模高效基因筛选，将之应用于肝脏cDNA文库的小规模的验证性研究得到了相当数量的已知基因、已知EST和一个新基因克隆；结合肝癌表达谱检测进行了肝癌相关基因的差异表达研究。阵列文库消减杂交结合体内染色质免疫沉淀可以进行全基因组尺度内的转录因子调控序列检测，我们进行了较小规模的验证确定了这一平台的有效性和实用性，进行了肝癌细胞经丝裂霉素处理后核转录因子NFкB的表达和结合位点检测，获得NFкB在此状态下的结合位点序列克隆的阵列文库，得到一些新的受NFкB调控的下游基因的启动子结合序列。
第一部分 阵列文库及阵列文库消减杂交用于肝癌相关基因筛选和差异表达的研究
利用平均插入子大小1.60kb的真核表达载体pcDNA3.1正常肝脏质粒文库制备阵列文库，利用BioRobotics TAS多功能基因芯片点样仪进行肝脏阵列文库消减杂交。利用阵列文库和经消减杂交筛选的基因制备基因芯片用于肝癌表达谱检测。制备了大鼠原发性肝癌模型用于基因表达检测。
结果显示，肝脏阵列文库消减杂交得到了相当数量的已知基因和一个新基因片段，利用单链环化反向巢式PCR进行5’RACE得到一个极低丰度的新基因
搏士学位论文 阵列文库及阵列文库消减杂交用于 中文摘要
——
拙W。肝癌表达谱基因芯片检测发现一个肝癌中相对癌旁组织特异性高表达的
mxr7基因和肝癌中不表达的CypZel基因。大鼠原发性肝癌模型模拟了从肝脏化
学性炎性损害、肝硬化、肝癌早期和肝癌晚期的各个阶段。mxr 7基因的大鼠同
源基因OCi－5在正常大鼠正常肝脏中表达极低，在大鼠肝癌组织中OCi－5表达急
剧升高，随着肝癌的进展表达逐渐上升，在肝硬化阶段OCi－5的表达高于正常肝
脏，在整个肝癌的发生发展过程中oci巧的表达是逐渐升高的。CypZel基因在人
肝硬化中的表达低于正常肝脏，而在肝癌中则几乎是完全不表达，在大鼠肝癌发
生发展过程的变化同OCi．5相反，表达明显下降直至几乎完全不表达。
结果提示，阵列文库消减杂交的方法可以高效筛选CDNA文库，得到文库中
的所有基因克隆。可以用极低的人力物力和财力达到同整个文库随机测序相同的
结果，利用阵列文库消减杂交从某种特定的生物、组织或细胞的CDNA文库中
筛选全部表达基因具有极大的应用价值，不但可以应用于本项目的肝脏和肝癌文
库筛选，还可以广泛地应用于其他领域的基因克隆工作。小规模的验证性研究中
得到了一个新基因m丁，这个基因是一个低表达基因，正在进行进一步的研究。
mxr7的大鼠同源基因oci－5基因同肝癌的演化关系密切，并且在肝癌早期已经发
挥其相应的功能，在肝癌的癌前病变时期即肝脏的炎性损害阶段和肝硬化阶段的
表达说明OCi－j基因表达上调在肝癌发生之前尤其是严重肝硬化时就己经发生，
提示OCi－5基因不但可以作为肝癌的诊断指标，也可以作为肝癌癌前病变的指标，
能够在肝硬化时期作出肝癌高危人群的预警。CypZel在肝癌细胞中可能是完全关
闭的，并且也随着肝硬化和肝癌的进展而逐渐降低。mxr沏dociJ基因和
CypZel基因与肝硬化和肝癌的发生发展密切相关，在肝癌的演化和进展过程中起
重要作用，并且提示可能会具有重要的诊断和理论研究价值。
第二部分 阵列文库消减杂交结合染色质免疫沉淀方法用于基因转录调控的
研究
转录调控是基因功能研究中的重点，转录因子对靶基因的结合和调控是转
录调控中的主要研究内容，染色质免疫沉淀方法可以进行体内转录因子的结合序
列的状态与变化的检测的新的强有力工具。结合我们的阵列文库消减杂交技术可
以在包括人类在内的复杂基因组中进行全基因组尺度内的大规模转录调控序列
的研究。我们建立了这样一个研究平台，进行了较小规模的验证确定这一平台的
有效性和实用性，同时进行了肝癌细胞 SK－HEP经丝裂霉素刺激后 NF出的表
达和结合位点检测，从而探讨其在肝癌化疗药物耐药中的作用，得到一些NFth
在此状态下的结合位点和受调控的下游基因。
结果显示，50冷ml丝裂霉素使 NF。B进入细胞核，而且在 8 ／J’时细胞内
5
博士学位论文 阵列文库及阵列文库消减杂交用于 中文摘要
肝癌相关基因筛选、表达和转录调控的初步研究
NF。B总量增加，提示 NF。B自身的表达增加。Western Blot检测丝裂霉素处
理后不同时间内总蛋白中的 NF K B含量升至较高水平，RTPCR方法检测 NF。
B的基因表达
Hepatocellular carcinoma (HCC) is one of the most popular and important malignant neoplasm in China. To elucidate the molecular mechanism of the proliferation, differentiation and transforming of the liver cell is very helpful for the early diagnosis and efficient treatment of the HCC. In present study we combined DNA microarray and inhibition subtract hybridization to set up a powerful technique named Arrayed Library Subtraction Hybridization (ALSH) to investigate gene screening, expression and transcription regulation of hepatocellular carcinoma associated gene. We use the ALSH to large-scale gene screening of liver cDNA library and harvest a number of known genes, ESTs and a new gene. We used the ALSH in the analysis of HCC associated gene expression profile. We also combined the ALSH and the chromatin immunoprecipitation (ChIP) to study the gene transcription regulation in the scale of whole genome and harvested an arrayed library of DNA fragment immunoselected by anti- NFicB antibody from liver cells tr
eated by mitomycin-C (MMC).
PART I Investigation of gene screening and differentiate expression profiling of HCC associated gene using arrayed library and arrayed library subtraction hybridization
The arrayed library was prepared from normal liver cDNA library purchased from Invitrogen company. Arrayed library subtraction hybridization was carried by the Biorobotics TAS multifuncton DNA microarray machine. The gene chip from the arrayed library and the genes screened from the arrayed library by ALSH were used to HCC expression profile analysis. The rat HCC and cirrhosis model were induced by DENA and TAA to further study of gene expression.
A number of known genes and ESTs as well as a new gene fragment were screened. The full length of new gene named as myl was cloned by the method of single strain reverse nested PCR. The mxr7/gpc3 gene, which is an important member of glypican family, was found as an overexpressed gene in the HCC by the method of expression profile DNA microarray. The cyp2el gene, one memeber of cytochrome P450 family,
was identified as a gene, which express highly and uniquely in. normal liver but do not express in HOC at all. The result was confirmed by the method of half-quantity HT-PCR and Northern Blot analysis. All the stages of occuireneee and development of the HOC was repeated in the chemical, induced model in. rat. The oci-5 gene hontologene of mxr7/gpc3 in rat, expressed hardly in normal not liver and. gradualy highly during the process of the development of HCC. Until the later phase of HCC oci-5 expressed very highly. The cyp2el gene expressed lower in the cirrhosis tissue than 'that: of normal liver tissue md did .not express in, HCC tissue. The change of the expression level of cyp2el during the development of HCC was opposite to that of mxr7/gpc3 gene.
It was showed that the ALSH technique could screen the cDNA library to get all the grates in 'the library just as- the achievement of sequencing of whole library, We have cloned a new gene my1 through small scale test experiment and identified as a low expression gene. The mxr7/gpc3/oci-5 gene was found to be closely associated to the accuroence and development of HCC and may play a key role in. the early stage before the occurrence of HCC, It was suggested that mxr7/gpe3/oci-5 gene may not only be a diagnosis index of HCC but also the index, in the stage of cirrhosis to anticipate the occrareoce of HCC. The cyp2el gene slopped to express in HCC and express gradually low during the development of cirrhosis and HOC.
PART II Combine the ALSH and ChIP to investigate the
transcription regulation in genome-wide scale
The specific site of DMA binding and activation of Izasnscriptionai factor are key process wf gene expression regulation. Chcomotin. immunoprecipitatioii (ChIP)
procedure was a powerful teeMiqu* to study the transcription factor prptdn-DNA ineractions in vivo, ChIP combined with ALSH could explore genome-wide searching of transcription factor spciilc DMA binding sites in the h
第一部分 阵列文库及阵列文库消减杂交用于肝癌相关基因筛选和差异表达的研究
利用平均插入子大小1.60kb的真核表达载体pcDNA3.1正常肝脏质粒文库制备阵列文库，利用BioRobotics TAS多功能基因芯片点样仪进行肝脏阵列文库消减杂交。利用阵列文库和经消减杂交筛选的基因制备基因芯片用于肝癌表达谱检测。制备了大鼠原发性肝癌模型用于基因表达检测。
结果显示，肝脏阵列文库消减杂交得到了相当数量的已知基因和一个新基因片段，利用单链环化反向巢式PCR进行5’RACE得到一个极低丰度的新基因
搏士学位论文 阵列文库及阵列文库消减杂交用于 中文摘要
——
拙W。肝癌表达谱基因芯片检测发现一个肝癌中相对癌旁组织特异性高表达的
mxr7基因和肝癌中不表达的CypZel基因。大鼠原发性肝癌模型模拟了从肝脏化
学性炎性损害、肝硬化、肝癌早期和肝癌晚期的各个阶段。mxr 7基因的大鼠同
源基因OCi－5在正常大鼠正常肝脏中表达极低，在大鼠肝癌组织中OCi－5表达急
剧升高，随着肝癌的进展表达逐渐上升，在肝硬化阶段OCi－5的表达高于正常肝
脏，在整个肝癌的发生发展过程中oci巧的表达是逐渐升高的。CypZel基因在人
肝硬化中的表达低于正常肝脏，而在肝癌中则几乎是完全不表达，在大鼠肝癌发
生发展过程的变化同OCi．5相反，表达明显下降直至几乎完全不表达。
结果提示，阵列文库消减杂交的方法可以高效筛选CDNA文库，得到文库中
的所有基因克隆。可以用极低的人力物力和财力达到同整个文库随机测序相同的
结果，利用阵列文库消减杂交从某种特定的生物、组织或细胞的CDNA文库中
筛选全部表达基因具有极大的应用价值，不但可以应用于本项目的肝脏和肝癌文
库筛选，还可以广泛地应用于其他领域的基因克隆工作。小规模的验证性研究中
得到了一个新基因m丁，这个基因是一个低表达基因，正在进行进一步的研究。
mxr7的大鼠同源基因oci－5基因同肝癌的演化关系密切，并且在肝癌早期已经发
挥其相应的功能，在肝癌的癌前病变时期即肝脏的炎性损害阶段和肝硬化阶段的
表达说明OCi－j基因表达上调在肝癌发生之前尤其是严重肝硬化时就己经发生，
提示OCi－5基因不但可以作为肝癌的诊断指标，也可以作为肝癌癌前病变的指标，
能够在肝硬化时期作出肝癌高危人群的预警。CypZel在肝癌细胞中可能是完全关
闭的，并且也随着肝硬化和肝癌的进展而逐渐降低。mxr沏dociJ基因和
CypZel基因与肝硬化和肝癌的发生发展密切相关，在肝癌的演化和进展过程中起
重要作用，并且提示可能会具有重要的诊断和理论研究价值。
第二部分 阵列文库消减杂交结合染色质免疫沉淀方法用于基因转录调控的
研究
转录调控是基因功能研究中的重点，转录因子对靶基因的结合和调控是转
录调控中的主要研究内容，染色质免疫沉淀方法可以进行体内转录因子的结合序
列的状态与变化的检测的新的强有力工具。结合我们的阵列文库消减杂交技术可
以在包括人类在内的复杂基因组中进行全基因组尺度内的大规模转录调控序列
的研究。我们建立了这样一个研究平台，进行了较小规模的验证确定这一平台的
有效性和实用性，同时进行了肝癌细胞 SK－HEP经丝裂霉素刺激后 NF出的表
达和结合位点检测，从而探讨其在肝癌化疗药物耐药中的作用，得到一些NFth
在此状态下的结合位点和受调控的下游基因。
结果显示，50冷ml丝裂霉素使 NF。B进入细胞核，而且在 8 ／J’时细胞内
5
博士学位论文 阵列文库及阵列文库消减杂交用于 中文摘要
肝癌相关基因筛选、表达和转录调控的初步研究
NF。B总量增加，提示 NF。B自身的表达增加。Western Blot检测丝裂霉素处
理后不同时间内总蛋白中的 NF K B含量升至较高水平，RTPCR方法检测 NF。
B的基因表达
Hepatocellular carcinoma (HCC) is one of the most popular and important malignant neoplasm in China. To elucidate the molecular mechanism of the proliferation, differentiation and transforming of the liver cell is very helpful for the early diagnosis and efficient treatment of the HCC. In present study we combined DNA microarray and inhibition subtract hybridization to set up a powerful technique named Arrayed Library Subtraction Hybridization (ALSH) to investigate gene screening, expression and transcription regulation of hepatocellular carcinoma associated gene. We use the ALSH to large-scale gene screening of liver cDNA library and harvest a number of known genes, ESTs and a new gene. We used the ALSH in the analysis of HCC associated gene expression profile. We also combined the ALSH and the chromatin immunoprecipitation (ChIP) to study the gene transcription regulation in the scale of whole genome and harvested an arrayed library of DNA fragment immunoselected by anti- NFicB antibody from liver cells tr
eated by mitomycin-C (MMC).
PART I Investigation of gene screening and differentiate expression profiling of HCC associated gene using arrayed library and arrayed library subtraction hybridization
The arrayed library was prepared from normal liver cDNA library purchased from Invitrogen company. Arrayed library subtraction hybridization was carried by the Biorobotics TAS multifuncton DNA microarray machine. The gene chip from the arrayed library and the genes screened from the arrayed library by ALSH were used to HCC expression profile analysis. The rat HCC and cirrhosis model were induced by DENA and TAA to further study of gene expression.
A number of known genes and ESTs as well as a new gene fragment were screened. The full length of new gene named as myl was cloned by the method of single strain reverse nested PCR. The mxr7/gpc3 gene, which is an important member of glypican family, was found as an overexpressed gene in the HCC by the method of expression profile DNA microarray. The cyp2el gene, one memeber of cytochrome P450 family,
was identified as a gene, which express highly and uniquely in. normal liver but do not express in HOC at all. The result was confirmed by the method of half-quantity HT-PCR and Northern Blot analysis. All the stages of occuireneee and development of the HOC was repeated in the chemical, induced model in. rat. The oci-5 gene hontologene of mxr7/gpc3 in rat, expressed hardly in normal not liver and. gradualy highly during the process of the development of HCC. Until the later phase of HCC oci-5 expressed very highly. The cyp2el gene expressed lower in the cirrhosis tissue than 'that: of normal liver tissue md did .not express in, HCC tissue. The change of the expression level of cyp2el during the development of HCC was opposite to that of mxr7/gpc3 gene.
It was showed that the ALSH technique could screen the cDNA library to get all the grates in 'the library just as- the achievement of sequencing of whole library, We have cloned a new gene my1 through small scale test experiment and identified as a low expression gene. The mxr7/gpc3/oci-5 gene was found to be closely associated to the accuroence and development of HCC and may play a key role in. the early stage before the occurrence of HCC, It was suggested that mxr7/gpe3/oci-5 gene may not only be a diagnosis index of HCC but also the index, in the stage of cirrhosis to anticipate the occrareoce of HCC. The cyp2el gene slopped to express in HCC and express gradually low during the development of cirrhosis and HOC.
PART II Combine the ALSH and ChIP to investigate the
transcription regulation in genome-wide scale
The specific site of DMA binding and activation of Izasnscriptionai factor are key process wf gene expression regulation. Chcomotin. immunoprecipitatioii (ChIP)
procedure was a powerful teeMiqu* to study the transcription factor prptdn-DNA ineractions in vivo, ChIP combined with ALSH could explore genome-wide searching of transcription factor spciilc DMA binding sites in the h
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