Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/53591
標題: 新肺癌轉移及腫瘤抑制基因,HLJ-1,之功能定性
Hlj1 Is a Novel Invasion and Tumor Suppressor
作者: 陳健尉
楊泮池
關鍵字: 基礎醫學類, 醫學技術, 生物技術, 臨床醫學類;tumor suppressor gene;腫瘤抑制基因;heat shock protein;non-small cell lung carcinoma;survival;metastasis;熱休克蛋白;非小細胞肺癌;存活;癌轉移;基礎研究
摘要: 
Background: Current clinico-pathologic prognostic variables may be inadequate in predicting relapse and clinical outcomes of non-small cell lung carcinoma (NSCLC). We have previously identified a panel of candidates of invasion-associated genes by microarray, including the HLJ1. Here, we investigated the clinical significance of HLJ1 in NSCLC and the role of HLJ1 in suppressing cancer progression. In addition, to investigate the transcriptional regulatory mechanism of HLJ1 gene expression, the identification and characterization of HLJ1 gene promoter/enhancer were performed.Methods: The differential expression of HLJ1 in cell lines was determined by microarray, real-time quantitative RT-PCR, Northern and Western blot analyses. We induced the over-expression of HLJ1 in highly invasive CL1-5 cells, and multiple assays were used to evaluate the function of HLJ1. The expression of HLJ1 in 71 NSCLC patients was measured by real-time quantitative RT-PCR and correlated with disease free and overall survival of the patients. We also Identified and characterized the HLJ1 downstream genes by microarray and pathway analysis, real-time quantitative RT-PCR and Western blot analyses. Flow cytometry was employed to monitor cell cycle progression. Report gene and electrophoretic mobility shift assays (EMSA) were used to characterize the minimal domain of HLJ1 enhancer. In addition, co-immuno-precipitation was employed to investigate the mechanism of transcriptional regulation on HLJ1. All statistical tests were two-sided.Results: HLJ1 is a significantly prognostic predictor of recurrence and overall survival in NSCLC patients. HLJ1 expression inhibits lung cancer cell proliferation, anchorage-independent growth, tumorigenesis, cell motility and invasion. HLJ1 can suppress cell cycle progression through a novel HLJ1/STAT1/ P21 WAF1 pathway and is p53 and interferon independent. The knockdown of HLJ1 expression by siRNA reverses this effect. Furthermore, we identified a novel enhancer segment at HLJ1 gene at -2,125 to -1,039 bp upstream to the transcription start-site. The 50-bp element at -1,492 to -1,443 bp is the minimal enhancer segment and the AP-1 site (-1,457 to -1,451) is the most important regulatory domain, where the FosB, JunB, and JunD can bind. We also identified a novel mechanism that up-regulates tumor suppressor HLJ1 expression by enhancer AP-1 binding with promoter YY1 and p300 through DNA bending and multi-protein complex formation. The combined expression of AP-1 and YY1 increase by more than five times the HLJ1-expression and inhibit in vitro cancer cell invasion.Conclusions: HLJ1 is a novel tumor suppressor in NSCLC, and high HLJ1 expression is associated with reduced cancer recurrence and prolonged survival of NSCLC patients. Our results also suggest that the transcription factors AP-1 at enhancer and YY1 at promoter can synergistically activate and up-regulate the tumor suppressor HLJ1.

研究目的: 目前的臨床病理預後因子並不足以完全預測非小細胞肺癌病人之術後復發及其後續之疾病進程。在先前的研究中,利用肺癌模式細胞株及基因微陣列法(microarray),篩選出許多與癌細胞侵入能力相關的基因,其中包括一個具潛力的基因HLJ1。此研究將調查HLJ1在非小細胞肺癌上的臨床重要性,及其在抑制癌症進程上所扮演的角色。此外,為研究HLJ1的轉錄調節機制,將進一步定性其最小促進子(enhancer)區域及相關的轉錄調節因子。研究方法: 此研究使用微陣列、即時定量RT-PCR、北方及西方墨點法來分析HLJ1於細胞株的表現量。轉染此基因至侵襲能力較高的細胞株(CL1-5),並分析其細胞增殖、試管內侵入、遷徙、細胞群聚及腫瘤形成能力。以即時定量RT-PCR分析71對正常及腫瘤組織中,HLJ1表現量與病人存活率的相關性。同時也利用微陣列、即時定量RT-PCR、西方墨點法來分析HLJ1的下游基因。流式細胞分析則用於細胞週期變化的研究上。報導基因及電泳移動偏移分析(EMSA) 則應用於最小促進子的定性分析上。此外,也使用免疫共沉澱法研究HLJ1的轉錄調控機制。所有統計分析均為雙尾測試。主要發現: 研究結果顯示,於非小細胞肺癌病人中,HLJ1是一個重要的疾病復發及存活率的預後預測因子。表現此基因會抑制肺癌細胞的增殖、細胞群聚、腫瘤形成、移動及侵入能力。其抑制或減緩細胞週期進行的可能作用機制,為經由ㄧ新的HLJ1/STAT1/P21 WAF1 調控途徑來達成,且不受p53及 interferon的影響。利用siRNA技術減少HLJ1表現則可逆轉此效應。已確認HLJ1促進子位於-2025至-1039核苷酸位置,其最小作 用區域則位於–1,492 to –1,443,主要包含SP1及AP-1轉錄調節因子結合序列,其中AP-1為決定因子,主要成員可能為FosB、JunD及JunB。將轉錄因子AP-1及YY1,個別、兩兩或三者同時,轉染至肺癌細胞株,均發現HLJ1蛋白質表現量增加,而細胞的侵襲能力則會降低。研究結果也進一步證實,促進子上的AP-1與啟動子上的YY1會經由DNA彎曲的機制(DNA bending),並藉由P300蛋白質的媒介,而形成蛋白質複合物,以進一步共同調節HLJ1的表現。結論: 於非小細胞肺癌中,HLJ1 是一個新的腫瘤抑制基因,表現HLJ1可降低肺癌的復發率並延長病人的存活率,是疾病復發及存活預後的重要預測因子。我們的研究結果也建議,在促進子上的轉錄因子AP-1及啟動子上的YY1可經由DNA彎曲機制與P300蛋白質形成複合物,進而協同活化HLJ1基因的表現。
URI: http://hdl.handle.net/11455/53591
其他識別: DOH95-TD-G-111-009
Appears in Collections:生命科學系所

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