透明細胞型腎細胞癌相關基因:血小板內皮細胞黏附分子I(PECAM-1)之功能性研究

Abstract

腎細胞癌是由腎小管上皮細胞病變所衍化而成的癌症，佔所有的惡性腫瘤中的百分之二，也是最致命的一種腎臟癌類。目前最常見的腎細胞癌類型是透明細胞型，而大部分的腎細胞癌對目前，因此目前為止手術根除是最主要的治療方式。然而五年的存活率仍低於百分之二十，除此之外臨床上還沒有可作為預測診斷或治療預後的分子指標。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，發現血小板內皮細胞黏附因子-1 f 這個基因在腎細胞癌中的表現有上升的情形。 PECAM-1 是一種細胞附著分子，一開始被發現於在血液中循環的血小板及白血球細胞表面之上，其主要功能是參與細胞和細胞間的連結。近年來發現PECAM-1除了原本的功能之外，還能作為訊息傳遞中轉接分子 (adaptor molecules) 的一個支架(scaffold) ，參與細胞內訊息傳遞。最近的研究指出PECAM-1在血管新生的過程中，可能扮演一個重要的角色，然而PECAM-1在血管新生及細胞癌化的過程中，所參與的完整分子機制至今還不是非常的清楚。 在本篇研究中我們發現了PECAM-1 的mRNA含量在臨床採集的腎細胞癌組織中有表現量上升的情形。之後我們在轉染PECAM-1腎細胞癌的細胞株 (HEK293)中，利用了共軛焦顯微鏡觀察到PECAM-1在細胞中主要存在在細胞膜上。而在功能性的研究中，我們發現在HEK293細胞中過度表現PECAM-1會造成調控細胞週期G1/S時期的細胞週期素D1(CCND1)基因的mRNA及蛋白表現量的上升，同時細胞株轉殖PECAM-1時其生長速率也較快。除此之外我們還觀察到血管新生因子(VEGF-A)，會因為PECAM-1的過度表現而造成mRNA及蛋白含量的上昇，其細胞的移動速率增快也在傷口癒合測試中 (Wound healing assay) 及細胞穿透測試 (transwell assay) 中可觀察得到。綜合以上的結果，我們認為在腎細胞癌中過度表現的PECAM-1不但可以使得細胞比較有生長的優勢，同時也使得細胞處於較癌化的形態。

Renal cell carcinoma (RCC) was thought to be one of major types of human kidney cancers, and it is characterized by a high mortality rate and a high resistance to chemotherapeutic treatment as well as radiations. The surgery remains to be the main method of treatment. Thus, we try to identify the genes associated with RCC to investigate the molecular mechanism of RCC. 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, the platelet endothelial cell adhesion molecule (PECAM-1) was found to be up-reglulated significantly in ccRCC tissues. PECAM-1 is a cell adhesion molecule expressed on the surface of circulating platelets and leukocytes, and it was also shown to be a scaffold for signaling and adaptor molecules for signal transduction in cells. It was demonstrated recently that PECAM-1 is associated in angiogenesis, but the molecular mechanism remained unclear.These findings suggest that PECAM-1 might play a crucial role in tumor development and progression. In present study, the expression of PECAM-1 gene was found upregulated in mRNA level of ccRCC tissue pairs. Confocal microscopy studies showed that PECAM-1 localized in plasma membrane. By functional studies, we have demonstrated that overexpression of PECAM-1 in HEK293 cell line induced the upregulation of a G1/S cell cycle regulatory gene, CCND1, and it also enhances the proliferation rate of transfected cells. Furthermore, overexpression of PECAM-1 also upregulated mRNA and protein levels of anginogenesis factor VEGF-A and further promoted the migration ability of HEK293 cells. Taken together, the results suggested that overexpression of PECAM-1 in ccRCC could provide several cell growth advantages and tumorgenesis.