肝癌 Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-08-15; 12(8): 1785-1788
在线出版日期: 2004-08-15. doi: 10.11569/wcjd.v12.i8.1785
肝癌相关cDNA片段的快速克隆和表达
赵弘智, 梁平, 李靖, 李洪艳, 黄小兵
赵弘智, 梁平, 李靖, 李洪艳, 黄小兵, 中国人民解放军第三军医大学新桥医院肝胆外科 重庆市 400037
赵弘智, 男, 1972-10-15生, 山西省大同市人, 汉族, 2002年第三军医大学博士研究生, 主要从事肝癌及胆管癌方面的临床和实验研究.
基金项目: 国家自然科学基金资助项目, No. 30170424.
通讯作者: 李靖, 400037, 重庆市, 中国人民解放军第三军医大学新桥医院肝胆外科. xqyylijing@163.net
电话: 023-65384825
收稿日期: 2004-03-05
修回日期: 2004-03-20
接受日期: 2004-03-24
在线出版日期: 2004-08-15

目的: 克隆原发性肝癌相关新基因.

方法: 利用抑制消减杂交法(suppression subtractive hybridi-zation, SSH)已经发现了1条新的肝癌相关基因片断表达序列标签(EST), 长447 bp, 经Genebank检索, 90%无同源性. 在其保守序列区设计了2条用于3'Race扩增的寡聚核苷酸引物(3'GSP2: 5'-CGCATAGTACCAGTATCGACAAAGG-3', 3'NGSP2: 5'-TCCACATTACGGACCCGACGGATT-3'), 利用cDNA末端快速扩增法(RACE)进一步克隆该基因的全长cDNA序列. 人原发性肝癌细胞株HepG2, 体外传代培养, 培养基为RPMI1640培养基. 提取HepG2总RNA, 方法参照SV Total RNA Isolation System的说明进行. RACE法扩增采用Clontech公司的Smart TM Race cDNA Amplification Kit. 将3'RACE-PCR扩增的目的片段以Race自带的产物纯化试剂盒进行纯化、回收, 然后将其克隆到PMD 18-T Vector中, 提纯质粒后进行酶切鉴定, 确认质粒内有插入片段, 由宝生物工程(大连)有限公司协助完成测序. 将克隆所得cDNA片段用NCBI提供的BLASTN与GeneBank的dbEST、nr数据库进行同源性比较, 确认代表新基因的EST并且登录GeneBank (http://www.ncbi.nlm.nih.Gov/submission). 3例病理标本取自西南医院肝胆科, 病理证实均为原发性肝细胞肝癌, 分别提取肝癌及远端正常肝组织总RNA. 将酶切回收的克隆插入片段分别进行同位素标记获得cDNA探针, 利用Clontech公司的ExpressHybTM杂交液通过RNA印记法检测克隆片段在肝癌及正常肝组织中的表达, 方法参照ExpressHybTM Hybridization Solution user manual说明进行. 同时, 利用互联网的基因表达分析序列数据库(http://www.ncbi.nlm.nih.gov/SAGE)(series analysis of gene expression, SAGE)对基因的表达及其表达水平进行分析, 从而确定其组织的分布.

结果: 得到5条3'EST(694 447-3, 724 447-3, 697 447-3, 711 447-3, 692 447-3; 大小500-550 bp), 5条3'EST均已登录GeneBank(登录号: CK730344, CK730345, CK730346, CK730347, CK730348). 对其中2条带有poly-A尾的3'EST(694 447-3, 724 447-3), 进行序列分析后, 发现他们是代表新基因或不同剪接体的EST, 且具有共同的保守序列. RNA印记分析显示694 447-3, 724 447-3在3例肝癌组织中的表达强度明显高于对应的正常肝组织. 通过SAGE文库分析基因的表达谱, 发现694 447-3和724 447-3在神经系统肿瘤、结肠癌、胃癌、乳腺癌肿瘤文库的表达高于对应的正常组织文库.

结论: 克隆所得的2条带有poly-A尾的3'EST可能是新的肝癌多基因家族成员. 利用 RACE技术可以快速、高效的克隆疾病相关基因.

关键词: N/A
引文著录: 赵弘智, 梁平, 李靖, 李洪艳, 黄小兵. 肝癌相关cDNA片段的快速克隆和表达. 世界华人消化杂志 2004; 12(8): 1785-1788
Rapid cloning and expression of hepatocellular carcinoma associated cDNA fragments
Hong-Zhi Zhao, Ping Liang, Jing Li, Hong-Yan Li, Xiao-Bing Huang
Hong-Zhi Zhao, Ping Liang, Jing Li, Hong-Yan Li, Xiao-Bing Huang, Department of Hepatobiliary Surgery, Xinqiao Hospital, Third Military Medicial University, Chongqing 400037, China
Supported by: the National Natural Science Foundation of China, No. 30170424.
Correspondence to: Dr Jing Li, Department of Hepatobiliary Surgery, Xinqiao Hospital, Third Military Medicial University, Chongqing 400037, China. xqyylijing@163.net
Received: March 5, 2004
Revised: March 20, 2004
Accepted: March 24, 2004
Published online: August 15, 2004

AIM: To clone and characterize novel genes that might be involved in hepatocellular carcinoma.

METHODS: An EST fragment differentially expressed between hepatocellular carcinoma (HCC) and its adjacent nontumorous liver tissues had been cloned using suppression subtractive hybridization (SSH). With this approach, we identified a novel EST that consisted of 447bp. In order to gain its full-length cDNA fragment, two gene-specific primers were designed for 3'-rapid amplification of cDNA end RACE. One HCC cell line, HepG2, was maintained in the RPMI1640 media and recommended culture conditions. Total RNA was extracted from HepG2 by the SV Total RNA Isolation System (Promega). RACE reactions were prepared using the SMARTTM RACE cDNA amplification kit (Clontech). Initial amplification was carried out with gene-specific primer 3'GSP2(5'-CGCATAGT ACCAGTATCGACAAAGG-3'), followed by nested PCR using gene-specific primer 3'NGSP2(5'-TCCACATTACGGACCCGACGGATT-3'). These amplified cDNA fragments obtained from RACE were subcloned into the PMD18-T vector (TaKaRa) and sequenced by ABI PRISM377 DNA sequencer. Basic local aligment search tools were carried out using BLASTN and dbEST and nr database. Northern blot was applied to detect the expression of these cDNA fragments between HCC and its adjacent nontumorous liver tissues. A total of 3 liver specimens were collected from the Southwest Hospital of Chongqing in China. The final diagnosis of HCC was confirmed by histological examination. Total RNA was extracted from either HCC or its adjacent nontumorous liver tissues in the same way. These cDNA fragments were excised from the PMD18-T vector, purified and 32P-labeled as cDNA probes using the random primed labeling method. Northern blot was prepared by using the ExpressHybTM hybridization solution (Clontech) according to the protocol provided by the manufacturer. Combination of Northern blot and virtual Northern (http://www.ncbi.nlm.nih.gov/SAGE)(series analysis of gene expression, SAGE), expression of these cDNA fragments in multiple carcinoma and normal tissues were analyzed.

RESULTS: Five EST fragments (694 447-3, 724 447-3, 697 447-3, 711 447-3, 692 447-3) were cloned, including two EST fragments with ploy (A) tail (694 447-3, 724 447-3). Compared with ESTs in GeneBank, the two EST fragments with ploy (A) tail represented novel genes with a common sequence. Five EST fragments accession numbers in Genebank were CK730344, CK730345, CK730346, CK730347, CK730348, respectively. Northern blot revealed that 694 447-3, 724 447-3 presented higher expression in HCC than that in its adjacent nontumorous liver tissues. Virtual Northern blot (SAGE) revealed that 694 447-3, 724 447-3 presented higher expression in multiple cancers that contained in brain, colon, breast, lung and stomach than that in their normal tissues.

CONCLUSION: Two novel human hepatocellular carcinoma-associated cDNA fragments are identified. RACE technique is a rapid and effective method for seeking for disease related genes in specific tissues.

Key Words: N/A
0 引言

通过抑制消减杂交法(suppression subtractive hybridization, SSH)我们已经发现了1条新的肝癌相关基因片段EST[1], 长447 bp, 经Genebank检索, 90%无同源性. 在此基础上, 我们采用RACE技术, 从肝癌组织中钓取肝癌相关的多个cDNA片段[2-3].

1 材料和方法
1.1 材料

人原发性肝癌细胞株HepG2, 由西南医院消化科司遂海同学惠赠. 体外传代培养, 培养基为RPMI1640培养基(Hyclone公司), 内含抗生素及100 mL/L的小牛血清. 细胞总RNA提取采用Promega公司的SV Total RNA Isolation System. RACE法扩增采用Clontech公司的Smart TM Race cDNA Amplification Kit.

1.2 方法

肝癌细胞株HepG2总RNA的提取参照SV Total RNA Isolation System的说明进行[4]. Race法扩增采用Clontech公司的Smart TM Race cDNA Amplification Kit中介绍的方法[5-7], 严格按说明操作. 3'Race ready-cDNA: RNA 1 μL (3 μL)、3'-CDS 引物1 μL、ddH2O 1 μL, 终体积5 μL. 在PCR仪(Bio-Rad) 70 ℃孵育2 min, 冰上冷却2 min, 之后加入: 5× First -strand buffer 2 μL、DTT 1 μL、dNTP mix 1 μL、powerscript reverse transcriptase 1 μL, PCR仪42 ℃孵育1.5 h, Tricine-EDTA buffer稀释反应产物至100 μL, PCR仪72 ℃孵育 7 min. 以3'Race ready-cDNA为模板, 以试剂盒提供的通用引物为下游引物, 合成的3'Race引物为上游引物, PCR法扩增Race的3'末端. 在保守序列设计了2条用于3'Race扩增的寡聚核苷酸引物(3' GSP2, 3'NGSP2). 通用引物(UPM)序列为: long 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'(0.2 μmoL/L); short 5'-CTAATACGAC TCACTATAGGGC-3'(1 μmoL/L); 3'GSP2引物(序列为: 5'-CGCATAGTACCAGTATCGACAAAGG-3', 10 μmoL/L). 3'RACE-PCR循环参数为: 94 ℃ 30 s, 72 ℃ 3 min→5 cycles; 94 ℃ 30 s, 70 ℃ 30 s, 72 ℃ 3 min→5cycles; 94 ℃ 30 s, 68 ℃ 30 s, 72 ℃ 3 min→30 cycles. 将第1次的产物稀释后作为模板用3'NGSP2引物(序列为: 5'-TCCACATTACGGACC CGACGGATT-3', 10 μmoL/L)与巢式引物NUP(序列为: 5'-AAGCAGTGGT AACAACGCAGAGT-3', 10 μmoL/L) 进行第2次扩增, 条件为: 94 ℃ 30 s, 68 ℃ 30 s, 72 ℃ 3 min→20cycles. 5 μL的PCR产物于1.2%琼脂糖凝胶上电泳与DNA Marker(DL2000)比较判断产物的大小. 将3'RACE-PCR扩增的目的片段以Race自带的产物纯化试剂盒进行纯化、回收, 然后将其克隆到PMD 18-T Vector中(购于Takara公司), 转染入感受态DH5细菌, 经过X-gal/IPTG蓝白斑的筛选后, 挑取多于10个的白色菌落进行质粒扩增, 提纯质粒后进行酶切鉴定, 确认质粒内有插入片段, 采用PCR、四色荧光、双脱氧终止法, 应用PE公司的ABI Prism TM 377XL DNA Sequencer进行测序, 引物为载体上的通用测序引物F Prime(M13-47)序列, 由宝生物工程(大连)有限公司协助完成. 将测序所得5条cDNA片段用NCBI提供的BLASTN与GeneBank的dbEST、nr数据库进行同源性比较[8-9], 确认代表新基因的EST并且登录GeneBank(http://www.ncbi.nlm.nih.Gov/submission).3例病理标本取自西南医院肝胆科新鲜手术标本, 病理证实均为原发性肝细胞肝癌, 分别提取肝癌及远端正常肝组织总RNA, 具体方法同前[4]. RNA转膜、固定, 通过对酶切回收的克隆插入片段分别进行同位素标记获得cDNA探针(Promega公司), 利用Clontech公司的ExpressHybTM杂交液进行预杂交、杂交, 洗膜、曝光及显影[10-11].用试剂盒中提供的β-actin作为对照探针进行RNA印记, 操作方法同前.用Image J 软件扫描各转录本灰度, 用公式Mean A = log10(255/Mean grey)计算强度, 以Mean A作为量化指标, 分析各转录本的表达丰度. 利用互联网的基因表达分析序列数据库(http://www.ncbi.nlm.nih.gov/SAGE)(series analysis of gene expression, SAGE)对特定基因的表达及其表达水平进行分析, 从而确定其组织分布[12-13].

2 结果
2.1 3'RACE-PCR产物的序列

设计3'GSP2和3' NGSP2进行Race, 共得到5条大小不等的片段, 分别命名为694 447-3, 724 447-3, 697 447-3, 711 447-3, 692 447-3; 大小500-550 bp(图1, 2), 对扩增的重组质粒进行酶切鉴定(图3). 对这些序列进行分析, 有2条是带有poly(A)尾的cDNA的片段, 1条片段可见加尾信号AATAAA, 将这些序列在计算机软件上进行比对(align)发现; 除靠近5'端的共有序列外, 在3'端序列有明显的不同, 而且这些序列不能够互相的囊括, 提示可能是不同基因的3'端[14-15].

图1
图1 3'RACE-PCR产物. M: marker; A: 3'RACE-PCR.
图2
图2 3'RACE-nestPCR产物. M: marker; B: 3'RACE-nest-PCR.
图3
图3 酶切鉴定重组质粒. M: marker ; 1: 692 447-3; 2: 711 447-3; 3: 697 447-3; 4: 724 447-3; 5: 694 447-3; 6: 质粒对照.
2.2 cDNA片段核苷酸序列的同源性

在5个测序的克隆中, 与GenBank的nr数据库进行同源性的比较显示, 2条带有poly(A)尾的cDNA的片段(登录号为: CK730344, CK730345)与基因编码序列的同源性均小于50%, 因而相应的基因及功能均不清楚, 可能代表新的未知基因[16-20].5条3'EST于2004-2-16呈递GeneBank, 现已登录, 登录号: CK730344, CK730345, CK730346, CK730347, CK730348.RNA印迹(图4)显示2条cDNA(694 447-3和724 447-3)在3例肝癌组织中的表达强度高于其对应的正常肝组织.

图4
图4 3对肝癌及正常对照组织RNA印迹结果. N: normal; C: cance
2.3 基因电子表达谱分析

694 447-3和724 447-3在肿瘤和正常Sage文库中的表达, Sage文库总tags数(表1).

表1 cDNA在肿瘤和正常Sage文库中的基因电子表达谱.
ProbeSage libraries
Brian
Colon
Breast
Lung
stomach
NCNCNCNCNC
694 447-3154911200170000
724 447-3011000000019
N: normal; C: cancer.
3 讨论

原发性肝癌是常见的恶性肿瘤之一, 居恶性肿瘤死因的第2位, 且有逐年增高趋势[21-22].在肝癌的研究中[23-24], 虽然已经肯定了很多在肝癌中异常变化的基因, 对肝癌的诊断与治疗有一定的参考价值, 但他们最初并不是通过直接研究肝癌所得到, 所以从肝癌组织出发, 进行肝组织癌变过程中基因型变化的研究, 可以为明确肝癌的发生机制及早期诊断提供进一步的线索.

Race技术是一种快速、灵敏、短时间内即可以获得新基因cDNA全长序列的方法, 而且Race能够产生大量的独立克隆[25-26].我们在进行差异片段的克隆时发现, 用基因特异性的引物在肝癌组织中进行3'Race的扩增, 同时扩增出5条高度同源的ESTs的片段, 他们在5'端的一致性很好, 蛋白质的编码序列可能有高度的保守性, 但是3'端差异却很明显, 这与多基因家族的特点是吻合的, 而且癌基因与抑癌基因多以基因家族的形式出现[27-30].通过测序分析, 他们有2条是带有poly(A)尾的3'EST, 经用NCBI提供的BLASTN对GeneBank的nr数据库进行电子信息杂交, 这是代表新基因的EST, 已经在GeneBank上注册.

为了对得到的2条候选同源基因与肝癌的关系做进一步的确证, 使用RNA印记分析694 447-3, 724 447-3显示其在3例肝癌组织中的表达强度明显高于其对应的正常肝组织; 为了更全面的检测克隆所得到的基因在其他肿瘤中的表达丰度及对其功能进行预测, 通过SAGE文库来分析基因的表达谱, 表明694 447-3和724 447-3在肿瘤文库表达高于正常文库, 这些肿瘤文库来源于神经系统肿瘤、结肠癌、胃癌、乳腺癌, 结合他们在肝癌中的表达情况, 提示在肝癌的发生、发展中他们很可能是共同表达的新基因簇.

编辑: N/A

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