利用全長cDNA資料庫鑑定與肝癌相關之目標基因

肝癌是台灣發生率及死亡率最高的癌症之ㄧ，B型肝炎或C型肝炎的感染、黃趜毒素及酒精的攝取已經被證明直接與肝癌的發生有關。在致癌過程中，累積染色體及基因的變異會使得正常肝細胞轉型並逐漸惡化成為肝癌細胞，其中基因突變及基因表現量的改變在致肝癌機制中扮演了重要的角色。我們與日本東京大學菅野純一及鈴木驤博士合作建構肝癌及其周邊組織之全長cDNA資料庫，並利用高速自動化設備，完成了58,251個肝癌及其周邊正常組織之cDNA定序，並經過電腦進行基因比對，我們找到180個在肝癌中表現量上升及279個在肝癌中表現量下降的基因。根據其細胞功能作進一步的分類後，發現許多可能與肝癌的發生以及發展有密切的關係，這些基因及其表現量變化與其影響肝細胞之生理功能，包括：1,致癌基因；2,細胞凋亡；3,抑癌基因；4,訊息傳遞；5,細胞週期；6,血管新生等類型之基因。我們並利用即時定量PCR技術，在45對肝癌組織中定量分析165個基因的表現改變，其中102個基因有顯著的變化，其中又有26個基因被鑑定為與肝癌末期發展有關，我們利用phylogenetic的方法分析其中與早期及末期相關的61個基因，有八個基因群組被發現，其中第五到第八組又與肝癌末期關係較大，以此可以證明這些基因在肝癌進展中扮演非常重要的角色。此外，為了瞭解基因與病患死亡率的相關性，我們也利用Kaplan-Meier圖表進行分析，發現了14個基因與死亡可能有關而其中有10個基因亦與肝癌末期相關，這些良好的關聯性更證明我們可以利用這些方法，成功的鑑定出許多與肝癌進展有關的基因，希望藉此可以闡明這些基因與肝癌之發展，以開發肝癌之診斷方法及治療藥物。

關鍵字

肝癌；全長cDNA資料庫；致癌基因；抑癌基因

並列摘要

HCC (Hepatocellular carcinoma) is one of the most frequent cancers with high mortality rate in Taiwan and worldwide. During hepatocarcinogenesis, accumulation of chromosomal alternations and genetic mutations may cause the transformation of normal liver cells into malignant HCC cells, and gene expression changes are also playing important roles in this process. In order to identify the HCC-related genes, we cooperated with Drs. S. Sugano and Y. Suzuki in University of Tokyo and constructed the full-length cDNA library of HCC and its adjacent normal tissues. By using high-throughput DNA sequencer, 58,251 cDNA clones of HCC and its adjacent normal liver tissues were sequenced, and genes were identified by comparison with GeneBank (NCBI). Among them, 180 up-regulated and 279 down-regulated genes were found in HCC. From these genes, 165 genes were subjected to real-time quantitative PCR analysis (Q-PCR) in 45 pairs of HCC and their surrounding normal liver tissues, which may be correlated with hepatocarcinogenesis and tumor progression, and 102 genes with more than two-fold differential expression were identified, which were related to oncogenes, apoptosis, tumor suppressor gene, signal transduction, cell cycle, and angiogenesis. With clinicopathological data and statistical analysis, we revealed 26 genes to be strongly associated with advanced HCC, including VEGF, MMP9, RB1, etc. In addition, 61 genes with high differential expression and related to tumorigenesis were subjected to phylogenetic analysis, and with eight gene clusters were obtained related to tumor development. Among them, oncogenic and cell cycle genes were greatly contributed to early events of HCC development while genes involved in angiogenesis and antiapoptotisis were associated with late development. We also identified 14 genes significantly associated with patient survival. By pathway analysis, present study provides 3 molecular networks of HCC-associated genes, in which N-RAS, RAF, VEGF, and KNG were played central role in the network 1. These genes could be good candidates for the development of therapeutic and diagnostic targets.

並列關鍵字

Hepatocellular carcinoma ； Full-length cDNA library ； Oncogene ； tumor suppressor gene

參考文獻

Reference List

1. Buendia MA. Genetics of hepatocellular carcinoma. Semin Cancer Biol 2000;10:185-200.

2. Feitelson MA, Sun B, Satiroglu Tufan NL, Liu J, Pan J, Lian Z. Genetic mechanisms of hepatocarcinogenesis. Oncogene 2002;21:2593-2604.

3. Suriawinata A, Xu R. An update on the molecular genetics of hepatocellular carcinoma. Semin Liver Dis 2004;24:77-88.

4. Egner PA, Wang JB, Zhu YR, Zhang BC, Wu Y, Zhang QN, Qian GS, Kuang SY, Gange SJ, Jacobson LP, Helzlsouer KJ, Bailey GS, Groopman JD, Kensler TW. Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer. Proc Natl Acad Sci U S A 2001;98:14601-14606.

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利用全長cDNA資料庫鑑定與肝癌相關之目標基因

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