| 摘要: | 肺癌是世界各國癌症死亡的主要原因之一，台灣地區2002 年的統計每十萬個
男性中就有41.12 人死於肺癌，每十萬個女性中就有19.38 人死於肺癌。肺癌病
人若能在發生轉移前發現並治療其五年的存活率可以達到50-70%，一旦發生轉
移現象則五年存活率會小於5%。先前的研究中我們已經證實HLJ1 是一個抑癌
基因，並且在臨床檢體的分析中我們也發現HLJ1 基因可以有效的預測非小細胞
肺癌病人開刀後再復發以及低存活率的高危險群，是一個非常好的預後預測因
子。本實驗主要針對HLJ1 基因的生物功能以及可能參與的分子作用機制進行深
入探討。因此先建立HLJ1 基因細胞表現系統，將HLJ1 基因選殖至pCDNA3 表
現載體系統並轉殖至肺癌細胞株CL1-5 細胞內，以定量RT-PCR 和西方墨點法實
驗証明該細胞表現系統確實可以表現出HLJ1。利用HLJ1 基因細胞表現系統進
行基因功能分析，研究結果顯示在肺癌細胞中大量表現HLJ1 基因可以抑制細胞
的複製、移動以及侵入能力。在老鼠體內的腫瘤生長研究也發現HLJ1 基因可以
明顯的抑制腫瘤生長，此外也發現老鼠腫瘤中的血管新生會受到HLJ1 基因的抑
制。因此HLJ1 基因在細胞內可能參與多種分子調控機制，利用DNA 微陣列的
技術我們進一步分析HLJ1 基因可能參與的分子調控機制。DNA 微陣列研究結
果發現至少有1240 個基因的表現會受到HLJ1 基因所影響。我們進一步以定量
RT-PCR 驗證上述結果，確定HLJ1 基因會抑制SNAI2、HMGA2、NOLC1、CALD1
以及CD44 等基因的表現，此外HLJ1 基因會促進STAT1、p21WAF1, ISGF3G、
IFIT1、IFITM1、OAS3、G1P2、SERPINB1、TIMP3 以及TXNIP 等基因的表
現。這些受到HLJ1 基因所影響的基因已被證實參與了細胞生長周期、訊息傳遞、
血管新生以及細胞移動的作用機制，其中也包含一些轉錄因子和腫瘤抑制基因。
Lung cancer is the most common cause of cancer death in the world. In Taiwan in
2002, the mortality rate of lung cancer was 41.12 and 19.38 per 100,000 among men
and women, respectively. If lung cancer is diagnosed and treated before it metastasizes,
the five-year survival rate is approximately 50-70%. Once metastasis has occurred,
five-year survival rate drops to < 5%. Therefore, metastasis is the most critical
parameter determining patient survival from lung cancer. In previously studies, we
have demonstrated that HLJ1 is a tumor suppressor in NSCLC. Clinically, HLJ1 is also
a significantly prognostic predictor for recurrence and overall survival in NSCLC
patients, especially in patients with stage I disease. In this study, we focus on the
biological functions and the action mechanisms of the HLJ1. To investigate the
function of HLJ1, we transfected the pCDNA3-HLJ1 plasmid containing the
full-length of HLJ1 cDNA into lung adenocarcinoma cells (CL1-5), and evaluated the
HLJ1 expression using quantitative real-time RT-PCR and western blotting analyses.
The HLJ1 overexpressed transfectants of lung cancer cells exhibited a markedly
decrease in proliferation rate, anchorage independent growth, invasive and migratory
ability, and tumor growth in SCID mice. Furthermore, we also found that the
restoration of HLJ-1 expression could inhibition the angiogenesis in the tumor. The
function of HLJ-1 may serve as a tumor suppressor and suppressive HLJ-1 expression
is associated with aggressive tumor behavior in lung cancer. To investigate the
regulatory mechanism of HLJ1 on tumor suppression, the best way is to identify HLJ1
modulated downstream genes. We carried out Affymetrix oligonucleotide microarrays
to sort out the differentially expressed genes between HLJ1 restoration and control
cells. The SYBR Green real-time RT-PCR were used to validate the gene expression
changes observed by the array analysis. we found 1240 genes were altered at least
2-fold changes by HLJ1 restoration. The SNAI2, HMGA2, NOLC1,CALD1 and CD44
genes were suppressed by restoration of HLJ1 expression, whereas IRF1, STAT1, p21
WAF1, ISGF3G, IFIT1, IFITM1, OAS3, G1P2, SERPINB1, TIMP3 and TXNIP were
stimulated by HLJ1 expression. The HLJ1 modulated genes involved in broad range of
cell cycle, transcription factor, signal transduction, angiogenesis and other tumor
suppressor genes. |
