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肝癌HBV整合点附近Cyclin E1基因高表达的研究
王涛，王一，Sze Hang Lau，关新元261021 山东潍坊，解放军第89医院病理科(王涛)；上海市普陀区妇婴保健院病理科(王一)；香港大学临床肿瘤学系(Sze Hang Lau、关新元)
基金项目： 国家自然科学基金(30171046)
摘 要:目的 研究乙型肝炎病毒(HBV)整合肝癌基因组位点附近基因表达调控的改变。方法 先后应用Southern blot、基因组文库构建与杂交筛选技术，在1例肝癌组织中克隆、筛选与鉴定了2个HBV DNA在人基因组中的阳性整合克隆，并对全长序列进行了测序分析。采用Western blot方法检测病毒插入点附近基因Cyclin E1的表达。免疫组化方法检测Cyclin E1在由143例肝癌组织构成的组织芯片中的表达水平。结果 肝癌组织中有2条＞3.2 kb 的阳性杂交带，分别为4.5 kb和12 kb，并有2个不同的整合位点。测序分析表明，其中一个病毒整合点的两端均位于人染色体19q12，插入的片段长度为1108 bp，整合过程造成了2107 bp HBV DNA缺失。另一个病毒序列整合到染色体的5p13，插入的病毒序列长度为2587 bp，发生了基因重排。病毒插入位点附近Cyclin E1基因呈高表达。51.7%(74/143例)肝癌组织Cyclin E1表达阳性。结论 HBV DNA插入人基因组可能导致细胞Cyclin E1高表达，提示病毒整合人染色体的潜在致癌机制。
关键词:癌，肝细胞； 肝炎病毒，乙型； 基因文库； 蛋白质阵列分析； 细胞周期蛋白E
Cyclin E1 gene overexpression near to hepatitis B virus integrated site in hepatocelluar carcinoma
WANG Tao，WANG Yi，SZE Hang Lau，GUAN Xin-yuan.Department of Pathology，Maternal and Infant Health Hospital，Putuo District，Shanghai 200060，China
Abstract:Objective To study the genomic regulating affections of hepatitis B virus (HBV) integrated into human genome of hepatocellular carcinoma (HCC).Methods The two HBV positive integrants were cloned and screened and identified in patient′s tissues with HCC by Southern blot and construction of genomic DNA libraries and library screening，respectively.The isolated genomic DNA fragment containing HBV integrant was analyzed by direct sequencing using the automatic sequencer.The expression of Cyclin E1 close to HBV inserted site in chromosome was detected by Western blot，and as well as in tissue microarray consisted of 143 HCC samples by immunohistochemistry.Results The 4.5 kb and 12 kb of two positive hybridized lanes larger than 3.2 kb which had the different integrated sites were identified.The sequencing analysis demonstrated that one of integrants mapped to chromosome 19q12 with two junction sequences.The length of inserted fragment was 1108 bp and cause the 2107 bp deletion of HBV DNA.Another one of the integrants located to chromosome 5p13 region and the viral fragment was 2587 bp accompanied by gene rearrangments.Cyclin E1 gene near to the HBV integrated site demonstrated the overexpression，and 51.7% (74/143 cases) overexpression in HCC tissue microarray as well.ConclusionsCyclin E1 overexpression in HCC tissue may be caused by HBV inserted into human genome and suggested that the latency mechanism of the viral integration.
Key words:Carcinoma，hepatocellular； Hepatitis B virus； Gene library； Protein array analysis； Cyclin E
文献标引:王涛，王一，Sze Hang Lau，关新元.肝癌HBV整合点附近Cyclin E1基因高表达的研究[J/CD].中华临床医师杂志：电子版,2010,4(7):949-953.
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