Insight into hepatocellular carcinogenesis by quantitative analysis of differential expressed genes in hepatocellular carcinoma

肝細胞癌是國人癌症死因的第一位。其發生的主要原因與B 型肝炎病毒、C 型 肝炎病毒的慢性感染有關，其他可能的因素包括食物遭黃麴毒素的污染或遺傳缺 陷，然而真正的分子致病機轉目前尚不清楚，有待進一步的研究。 之前我們利用Incyte Human GEM 1 cDNA 基因晶片（約9700 基因）來分析肝 細胞癌的 mRNA 轉錄輪廓(transcription profile)，比較肝癌病人非腫瘤和腫瘤組織之 間差異表現的基因。總共找到73 個候選基因在肝腫瘤組織表現量上升，58 個候選 基因表現量下降。我們將基因晶片結果中找到的差異性表現基因，比對已發表過的 LOH 及CGH 實驗結果，我們挑選出3 個基因(11 β-hydroxysteroid dehydrogenase 、 histidine-rich glycoprotein 、CCT-γ chaperonin)作進一步的實驗。首先我們抽取了44 位肝細胞癌病人的肝癌組織以及其周圍非肝癌組織的RNA ，將mRNA 反轉錄成 cDNA 。為了確認cDNA 基因晶片實驗結果的正確性，即時定量PCR （real-time quantitative PCR ）系統被用來評估這3 個基因的mRNA 差異表現量是否有一致性。 實驗結果發現，即時定量PCR 結果與晶片數據有高度相似性。 另外我們也檢視這3 個基因在肝癌病人族群表現情形，在55%（25/44 ）的HCC 病人，其周圍非肝癌組織中HSD11 mRNA 含量大於肝癌組織中兩倍以上。HRG 亦 發現在59%(26/44)病人癌組織中有表現下降情形。而Cctg 則是有59%（26/44 ）的 病人，其肝癌組織RNA 含量是大於周圍非肝癌組織的兩倍以上。即時定量PCR 系 統提供一個大量快速篩檢平台來確認個別基因表現，將可輔助基因晶片技術。我們 將更進一步確認這些基因的蛋白表現情形，希望可以對肝癌的致病機轉有更多的了 解，進而發現可以當作診斷或是治療標的基因。Hepatocellular carcinoma (HCC) is one of the most common cancer in the world and is the leading cause of cancer death in Taiwan. Chronic hepatitis B and recently the hepatitis C viral infection are thought related to the development of HCC. However, the basic molecular mechanism remained to be clarified. In 2001, we have analyzed the transcriptional profiles of six HCC specimens using Incyte Human GEM1 cDNA microarray which consists of ~9700 genes. Comparison of expression profiles between HCC tumors and the corresponding nontumor tissues, we identified 73 genes were up-regulated and 58 genes were down-regulated in tumor tissues. The highly concordant overexpressed profile included transcripts involved in the function of respiration control, protein synthesis, degradation, cytoskeleton and carcinogenesis. Down-regulation of genes related to drug metabolism, differentiation and immune response was observed in the majority of the tumors examined. After comparing the LOH, CGH, and microarray data, we pick 3 differential expression genes for further analysis. First, we extracted 44 pair of HCC RNA from tumor part and corresponding nontumor part. RNA reverse transcribed to cDNA. In order to validate cDNA microarray data, real-time quantitative polymerase chain reaction (QPCR) was used to evaluate 3 gene (11 β-hydroxysteroid dehydrogenase, histidine-rich glycoprotein and chaperonin CCT-γ) expressions. Highly correlation was observed between these two methods. We also screen these 3 gene expression levels in a panel of 44 HCC specimens. 11 β-HSD and HRG were down-regulated in 55% (25/44), and 59% (26/44) HCC cases respectively. CCT-γwas up-regulated in 59% (26/44) HCC cases. The expression profiles of the 6 HCC cases provide valuable information for elucidation of hepatocarcinogenesis, and also represent the potential biomarkers implicated in this malignancy