NORPEG基因高度表現於C型肝炎病毒蛋白質表現細胞之研究

Up-regulation of NORPEG Gene in Hepatitis C Virus Proteins Expressing Cells

李佳穎(Jia-Ying Lee)

指導教授 ： 張明富

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02456

摘要

肝細胞癌(HCC)是世界上最普遍的癌症之一，儘管在最近幾年中陸續發展了新的治療方式，不過肝癌之預後效果還是不太理想。造成肝癌之主要因素為由B型肝炎病毒(HBV)以及C型肝炎病毒(HCV)所引起之慢性肝炎，以及其他不同之致癌物包括了黃麴毒素等。本篇論文主要是在探討C型肝炎病毒以及肝癌之間的關係。
最近研究指出受到C型肝炎病毒感染之B細胞淋巴癌其p53基因會出現高度之突變情形。為了瞭解與肝癌生成有關之分子機制，本論文利用人類肝癌細胞株Huh7以及經繼代培養後含有HCV subgenomic replicon之Huh7細胞株，來觀察其p53基因之變異情形。在分析過Huh7以及第12、22、47、80和132代HCV Replicon細胞之p53基因後，我們觀察到了兩個點突變的位置。其一為第411組核苷酸對C•G突變為T•A，該突變沒有造成該位置胺基酸的改變 (Leu)。另一個為第793組核苷酸對A•T突變為G•C，該突變造成了p53蛋白質第220個胺基酸Tyr轉變為Cys。而於其他含有HCV subgenomic replicon的Huh7細胞中並沒有觀察到p53基因上更多的突變。
另外在本實驗室DNA微陣列分析的結果中，發現一個未知功能的NORPEG基因，其表現於第33代HCV Replicon細胞中有提升的現象。同時在之前的研究中亦指出該基因於HCC中的表現也有提升的情形。經由即時定量聚合酶連鎖反應的分析得到該基因在第12及22代HCV Replicon細胞中的表現出現了增加的現象。接著利用Western blot analysis探討了NORPEG蛋白質於不同細胞株中的表現情形，發現其於肝癌細胞株中(Huh7,HepG2)之表現較子宮頸癌細胞株(HeLa)為多，並且於穩定表現HCV核心蛋白質之HeLa細胞(Core-6, Core-13)中的表現也較HeLa細胞為多。
由以上實驗顯示，與HCV非結構性蛋白質相較之下，核心蛋白質對於NORPEG蛋白質之表現產生了較多的影響。HCV核心蛋白質以及非結構性蛋白質如何調控NORPEG基因，以及NOPPEG蛋白質可能參與肝癌生成之分子機制仍待進一步之研究來闡明。

關鍵字

C型肝炎病毒 ； 肝癌

並列摘要

Primary hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. Despite the development of novel therapeutic methods in recent years, prognosis of advanced HCC is still poor. Major risk factors for HCC are chronic hepatitis resulting from infection with hepatitis B or C virus, and exposure to various exogenous carcinogens including aflatoxin B1. In this study, the relationship between HCV and HCC is under investigation.
Recent studies identified hypermutation of p53 gene in HCV-infected B cell lymphoma. To understand the molecular mechanisms of HCV involved in the hepatocarcinogenesis, mutation status of p53 in HCV replicon-containing cell line and the parental Huh7 cells were analyzed. Two mutations were found in the p53 gene of Huh7 and HCV replicon cells continually passaged for 12, 22, 47, 80 and 132 generations. C to T mutation at nucleotide 411 resulted in a silent mutation, whereas the A to G mutation at nucleotide 793 caused a tyrosine to cysteine mutation at amino acid residue 220. No additional mutations were observed in the HCV replicon cell lines.
On the other hand, by performing DNA microarray analysis, our laboratory have previously identified a novel gene NORPEG that was up-regulated in the HCV replicon-containing Huh7 cell line (the 33rd passage). This result was confirmed by real-time PCR analysis of HCV replicon cell lines (the 12th and the 22nd passages). NORPEG gene was recently reported to be up-regulated in HCC, but its function is still not fully understood. Antibodies specific to NORPEG protein were generated. Western blot analysis demonstrated that the expression levels of NORPEG protein were higher in the human hepatoma cell lines Huh7 and HepG2 than in non-hepatic HeLa cells. In addition, the expression level of NORPEG protein was also higher in HCV core protein-expressing HeLa cells (Core-6 and Core-13) when compared to the parental HeLa cells. These results suggest that NORPEG may have a role involved in the pathogenesis of the core protein and the hepatocarcinigenesis of HCV.

並列關鍵字

p53 ； NORPEG ； hepatitis C virus ； hepatocellular carcinoma

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