腎細胞癌相關基因Heme oxygenase-1與Annexin A2之功能性研究

Studies on the function of RCC-associated genes, Heme oxygenase-1 and Annexin A2

賴彥翰(Yen-Han Lai)

指導教授 ： 林榮耀

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01745

摘要

腎細胞癌佔所有的惡性腫瘤中的百分之二十，也是最致命的一種腎臟癌類型。目前最常見的腎細胞癌類型是透明細胞型，它對於傳統的治療方式具有高度的抗性，所以手術切除是目前主要的治療方式。因此，我們希望藉由研究腎細胞癌相關基因來了解腎細胞癌形成的分子機制。在先前的研究中，本實驗室的唐賽文先生建構了腎細胞癌以及正常腎臟組織的全長cDNA庫，希望藉由比較兩者之間不同基因的表現來找出與腎細胞癌形成的相關基因。經過比較之後，發現Annexin A2 (AnxA2)和Heme oxygenase-1 (HO-1)這兩個基因在腎細胞癌中的表現有上升的情形。
AnxA2是一個由鈣調節和磷酸脂鍵結的蛋白質，在許多的轉型的正常細胞株和癌細胞株中都有發現它的表現量上升的情形。HO-1是一個熱休克蛋白，它能被許多逆境因子誘發。它能催化血基質(heme)的分解，並產生一氧化碳(CO)、膽紅素(bilirubin)，和鐵離子(Fe2+)。HO-1在許多癌症中也都有表現量上升的情形，在某些癌症中則是和血管增生有關。這些結果顯示AnxA2和HO-1對於癌細胞的生長和發展中扮演了非常重要的角色。
在本篇研究中我們發現了AnxA2和HO-1的mRNA和蛋白質在臨床採集的腎細胞癌組織中有表現量上升的情形。而在免疫組織染色的實驗中，我們發現HO-1會進入細胞核。在腎細胞以及腎細胞癌的細胞株中，我們利用共軛焦顯微鏡以及細胞核分離法進一步確認HO-1也會進入細胞核。於是，我們使用酵母菌雙雜合篩選系統來尋找可能在細胞核中與HO-1作用的蛋白質。在功能性的研究中，我們發現在HEK293細胞中過度表現HO-1會造成調控細胞週期G1/S時期相關的蛋白的表現量的上升，另外它也能夠使得細胞能對抗cisplatin所引起的細胞凋亡並且造成bcl-2蛋白質的表現量上升。綜合以上的結果，我們認為在腎細胞癌中過度表現的HO-1不但可以使得細胞比較具有癌化的傾向，而且還能使得細胞對抗抗癌藥物所引起的細胞凋亡。另外，由酵母菌雙雜合篩選系統所找到與HO-1作用的蛋白質中，我們發現HO-1可能會參與細胞核中的訊息傳遞，並且也可能參與NFκB的活化。

關鍵字

腎細胞癌

並列摘要

Renal cell carcinoma (RCC) accounts for 2% of all adult malignancies and is the most lethal of common urologic cancers. The major type of RCC is clear cell RCC (ccRCC). It is highly resistant to traditional therapies, and surgery remains to be the main method of treatment. Therefore, identification of RCC-associated genes is important in understanding the molecular mechanisms of ccRCC. In previous studies, full-length enriched cDNA libraries of ccRCC and normal kidney tissues were constructed by Mr. Sai-Wen Tang from this laboratory. By comparing the differential gene-expression profiles of ccRCC and normal tissues, Heme oxygenase 1 (HO-1) and Annexin A2 (AnxA2) were found to be up-reglulated in ccRCC tissues.
AnxA2 is a Ca2+-dependent phospholipid binding protein, and its overexpression has been found in virally transformed cell lines and various tumors. HO-1 is a heat shock protein induced by a variety of stress stimuli. HO-1 catalyzes the degradation of heme to produce CO, biliverdin, and Fe2+. Elevated HO-1 expression was found in various tumors and linked to angiogenesis in some tumors. These findings suggest that HO-1 and AnxA2 might play a crucial role in tumor development and progression.
In present study, the expression of HO-1 and AnxA2 genes were found up-regulated in mRNA as well as protein levels of ccRCC tissue pairs. Immunohistochemical studies showed that HO-1 localized in the nucleus in ccRCC tissues, and this was further confirmed in kideney and RCC cell lines by confocal microscopy and subcellular fractionation. Therefore, yeast two-hybrid was performed to identify HO-1 interacting proteins in the nucleus. By functional studies, we have demonstrated that overexpression of HO-1 in HEK293 cell line induced the up-regulation of several G1/S cell cycle regulatory genes, and it also protected cells from cisplatin induced apoptosis and up-regulated protein levels of bcl-2. Taken together, it is suggested that overexpression of HO-1 in ccRCC could provide a selective advantage towards cell transformation, and protect cells from cisplatin- induced apoptosis. Furthermore, the identification of putative HO-1 binding proteins by yeast two-hybrid suggested that HO-1 might participate in cell signaling in the nucleus and also the activation of the NFκB pathway.

並列關鍵字

RCC ； HO-1 ； Annexin A2

參考文獻

1.Lam, J.S., et al., Role of molecular markers in the diagnosis and therapy of renal cell carcinoma. Urology, 2005. 66(5, Supplement 1): p. 1-9.

2.Jones, J., et al., Gene Signatures of Progression and Metastasis in Renal Cell Cancer. Clin Cancer Res, 2005. 11(16): p. 5730-5739.

3.Atkins, D., R. Lichtenfels, and B. Seliger, Heat shock proteins in renal cell carcinomas. Contrib Nephrol, 2005. 148: p. 35-56.

4.Tang, S.W., et al., Identification of differentially expressed genes in clear cell renal cell carcinoma by analysis of full-length enriched cDNA library. J Biomed Sci, 2006: p. 1-8.

5.Ozaki, T. and S. Sakiyama, Molecular cloning of rat calpactin I heavy-chain cDNA whose expression is induced in v-src-transformed rat culture cell lines. Oncogene, 1993. 8(6): p. 1707-10.