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|標題:||HLJ-1 Is a Novel Invasion and Tumor Suppressor
|關鍵字:||基礎研究;metastasis;基礎醫學類, 生物技術;癌轉移;微陣列;熱休克蛋白;癌細胞侵襲;啟動子;microarray;heat shock protein;invasion;promoter||摘要:||
Metastasis, the malignant cancer cells from their primary sites of origin to distant secondary sites within the body, is the multiple-step process requires the accumulation of altered expression of many different genes. By using a microarray and invasion/metastasis cell line model in a previous study, we identified the DnaJ-like heat shock protein 40, HLJ1. Northern blotting analyses showed that the HLJ1 mRNA expression level was negative correlation with invasive and metastatic capability in these cell lines. The similar results were obtained with real-time quantitative RT-PCR and Western blotting analyses. Furthermore, the reduced expression of HLJ1 was significantly associated with early postoperative relapse (P=0.0436) and shorter survival (P=0.0196) in adenocarcinoma patients. To determine whether HLJ1 expression is responsible for the tumor suppression or invasion suppression in lung cancer, we transfected the human HLJ1 cDNA into high invasive lung adenocarcinoma cells (CL1-5). After stable transfection of a HLJ1 cDNA expression vector, the proliferation of HLJ1 tranfectants decreased remarkably compared with the cells stably transfected with empty vector. The HLJ1 transfectants growth in soft agar and in SCID mice was markedly retarded. The cell invasion and migration assay also indicated that stable transfectants of HLJ1 in CL1-5 cells significantly suppressed the cell invasive and migratory ability compared with mock transfectants. In addition, we further characterize the putative promoter region of human HLJ1. The results indicated that the region from ？HHHV232 to +176 could drive the basal transcriptional activity of the HLJ1 gene. In this project, we will further characterize the effects of the putative factors on HLJ1 promoter regulation and attempt to identify its enhancer region. The interaction partners of HLJ-1 protein and the distribution of HLJ1 and its associated proteins in lung cancer cell will be also investigated. Furthermore, we will employ siRNA and microarray technologies to identify the downstream genes and evaluate the effect on cell cycle regulation when lost of HLJ1 function, which may assist us in exploring the putative mechanisms of HLJ1 involved in tumor/metastasis suppression and orchestrating the process of cancer metastasis.
癌轉移是指惡性腫瘤細胞在體內從初發生的位置移動到遠方第二個位置出現的過程，此過程涵蓋許多階段，需要許多不同的基因累積變異所致。在先前的研究中，藉由微陣列技術及肺癌轉移模式細胞株，我們發現HLJ1是一有潛力的腫瘤生長及轉移的抑制基因，此蛋白屬於DnaJ-like 40 kD熱休克蛋白家族的一員。北方墨點法、即時定量RT-PCR及西方墨點法分析結果顯示，HLJ1 mRNA及蛋白質的表現量和這些細胞株的侵襲╱轉移能力成反比。分析肺腺癌病人檢體也發現HLJ1 mRNA多表現在正常組織，在腫瘤細胞的低表現量與早期術後復發(P=0.0436)及低存活率(P=0.0196)有顯著相關。將HLJ1 cDNA轉殖到具高侵襲能力的肺腺癌細胞CL1-5中，其增生速率遠小於只轉殖空載體的細胞。HLJ1轉殖株在軟瓊脂和SCID小鼠身上所造成的細胞聚落及腫瘤生長，都明顯發生遲緩的現象。細胞侵襲和移動分析結果亦指出，HLJ1穩定轉殖株會有效地抑制癌細胞侵襲及移動的能力。此外，選殖並定性HLJ1基因可能的啟動子調控區域，發現位於-232到+176的區域片段具有啟動HLJ1基因的基礎轉錄能力。在此計畫中，我們將進一步研究可能的調控因子對啟動子的調節作用，並嘗試選殖促進子的調控區域以分析HLJ1的調控機制；同時也將篩選與HLJ1進行交互作用的蛋白分子，及其在細胞內產生作用的區域；進一步利用siRNA及微陣列技術研究其下游基因表現及對細胞週期的影響，以建構出HLJ1基因參與在腫瘤及癌轉移抑制中的可能機制。
|Appears in Collections:||生命科學系所|
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