抑癌基因HLJ1（Human Liver DnaJ-like protein）經由環境壓力刺激後的調控表現研究

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Title:	抑癌基因HLJ1（Human Liver DnaJ-like protein）經由環境壓力刺激後的調控表現研究 Studies of the stress stimulation：regulation the expression of tumor suppressor HLJ1
Authors:	王恩瑋 En-wei Wang
Contributors:	張竣凱蔡孟峯嘉南藥理科技大學：生物科技研究所
Keywords:	熱休克蛋白40 抑癌基因啟動子 HLJ1 HSP40 tumor suppressor promoter
Date:	2007
Issue Date:	2008-12-03 11:17:30 (UTC+8)
Abstract:	根據行政院衛生署2006年統計資料，惡性腫瘤為臺灣地區十大死因之首。而惡性腫瘤死亡者又以肺癌和肝癌為主要原因。本論文主要針對肺癌之抑癌相關基因特性進行研究。前人研究指出在人類肺腺癌細胞株中，HLJ1大量表現會抑制癌細胞的增生(proliferation)、侵入(invasion)和轉移 (migration)。此外臨床上研究亦發現肺癌中HLJ1高表現量的病人，癌症的複發率會降低並且可以增加病人的存活率。HLJ1已被証實是一個能抑制腫瘤生長並且能抑制癌細胞轉移的抑癌基因。從HLJ1基因序列分析顯示其含有熱休克蛋白40的J區域和G/F區域。因此釵h研究者認為HLJ1基因可能具有熱休克蛋白的特性，然而熱休克因子如何調控HLJ1，以及在釵h的環境因子刺激下如：熱休克反應(heat shock)、細胞密度(cell density)、無血清(serum deprivation)、酸鹼度(pH-mediated)和麩醯胺酸(glutamine)等刺激之下HLJ1基因特性尚未進行研究。因此本論文進一步探討肺腺癌細胞經由環境刺激時的HLJ1基因表現調控特性。進行環境刺激實驗後，由結果可知在熱休克反應(43℃，1小時；再經不同時間培養)利用即時定量反轉錄聚合酶連鎖反應法分析法分析HLJ1 mRNA的表現。分析後受熱休克處理與未受熱休克處理的CL1-0和CL1-5細胞在4小時時，HLJ1 mRNA的表現分別有1.8和2.5倍的差異。另外再藉由西方墨點法分析CL1-0和CL1-5受熱休克處理後HLJ1蛋白表現量，相同的也發現受熱休克處理後其HLJ1蛋白表現量較高。在CL1-0和CL1-5細胞密度(5×105、1×106、5×106培養48小時)實驗中，低細胞密度(5×105)與高細胞密度(5×106)刺激之下，高細胞密度HLJ1蛋白表現量明顯比低細胞密度HLJ1蛋白表現量高。在無血清(無血清培養72小時)實驗中，CL1-0和CL1-5細胞在無血清培養72小時後與含血清相較後，CL1-0和CL1-5細胞HLJ1 mRNA的表現分別有2.5和6倍的差異。在蛋白的表現量上，無血清培養其HLJ1蛋白表現量也較高。在酸鹼度(pH 6.4培養液培養6小時)實驗中的CL1-0和CL1-5 HLJ1 mRNA的表現分別有2和3倍的差異。蛋白的表現上，經酸性培養液刺激後HLJ1蛋白表現量亦有增加。最後，在含有麩麩醯胺酸(麩醯胺酸4、8mM培養後經熱休克43℃；1小時再經4小時培養) CL1-0和CL1-5細胞中，在麩醯胺酸4、8mM刺激後，HLJ1蛋白表現量亦有明顯增高。在環境刺激實驗中，可得知HLJ1基因會受環境刺激而使得其表現量增加。所以我們也進一步分析HLJ1的啟動子(promoter)區域，經由初步的分析後發現HLJ1的啟動子區域可能含有熱休克區域(heat shock element)，構築HLJ1啟動子區域408bp。尋找出HLJ1熱休克主要反應的區域。從實驗結果可知在啟動子長度408bp這段序列經由熱休克反應(43℃，1小時；再經小時4小時培養)後利用Luciferase reporter assay方法分析。由結果得知在CL1-5細胞經熱休克反應後，其Luciferase activity有3倍的差異。因此我們可確認HLJ1的確會受到熱休克調控。 Lung cancer is most common cause of cancer death in Taiwan. Human Liver DnaJ-like protein (HLJ1) is a tumor suppressor gene in non–small-cell lung cancer (NSCLC) and its highly expression of HLJ1 is associated with reduced cancer recurrence and prolonged survival of NSCLC patients. Recent studies have indicated that HLJ1 expressed in NSCLC cells would inhibit cell proliferation, anchorage-independent growth, cell motility, invasion, and tumorigenesis. The HLJ1 DNA sequence analysis showed that it contained the J and G/F domain sequence of the Heat-shock protein 40 (Hsp40) family members. The amino-acid sequencing revealed that HLJ1 might be a member of heat shock protein family. Yet it has not been investigated that how heat shock factor can regulate HLJ1 expression in response to many stresses, such as heat shock, serum deprivation, cell density, exposure to heavy metals, sodium arsenite, and oxidative stress. Then we observed that the expression of HLJ1 in CL1-0 and CL1-5 adenocarcinoma cells was responsive to heat shock stress, cell-density, serum deprivation, pH-mediated stimuli and glutamine treatment before heat shock stress. For heat shock, serum deprivation, glutamine treatment experiment, CL1-0 and CL1-5 cells were plated on different dish in different count of cell number, and incubated at diverse conditions. In order to determining the critical region in HLJ1 promoter involved in heat shock response, the promoter region of HLJ1 was cloned into pGL3-basic vector. The Luciferase reporter assay was performed to detect the promoter activity of HLJ1. Furthermore, we will analyze the putative heat shock factor element (HSE) of HLJ1 promoter and construct various deletions of HLJ1 promoter. These studies imply stresses such as heat shock, high cell density, serum deprivation, pH-mediated and glutamine cultures are important factors involved in the up-regulation of HLJ1 expression.
Relation:	校內校外均不公開
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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