探討ENO1之表達和腫瘤細胞轉移蛋白表現的關係

The relationship of ENO1 and signal proteins involved in metastasis

劉淑惠(Shu-Hui Liu)

指導教授 ： 楊沂淵

共同指導教授 ： 劉柯俊

臺北醫學大學/醫學院/醫學科學研究所/碩士(2007年)

摘要

已有文獻指出α-Enolase (ENO1)在某些人類癌症病患之腫瘤中其表達量之高低和腫瘤轉移及病患之存活有密切關係，例如：Lung cancer、Hepatocellular carcinoma (HCC)，在老鼠之黑色素細胞腫瘤(melanoma cell line)也觀察到類似結果。ENO1的overexpression可能藉由許多機轉，造成腫瘤細胞的侵犯和轉移。
本論文之研究目的分兩部份：第一部份，將以黑色素腫瘤細胞株分析ENO1表現與腫瘤細胞migration能力之關聯性、並調控ENO1表現以了解是否相對影響腫瘤細胞migration相關蛋白或其基因的表現。第二部份，將分析肝癌病人在藥物治療後不同時期體內對ENO1免疫反應之變化。
材料和方法：以高轉移之老鼠黑色素腫瘤細胞B16F1L5為材料，以short hairpin RNA(shRNA)方式建立ENO1 knockdown之細胞株，及一株ENO1 knockdown之對照細胞株(B16F1L5 pSM2 vector control)，進行下列實驗：(一)以in vitro slide wound-healing assay，探討降低ENO1表達是否影響腫瘤細胞之migration。(二)以RT-PCR分析調降ENO1是否影響migration 相關基因(uPA、uPAR、MMP-2、MMP-9、cdc42、RhoC) mRNA的表現。(三)收集腫瘤細胞蛋白，以western blotting analysis分析ENO1 expression和migration相關基因(MMP-2、MMP-9、c-Met、FAK、FAK(pY397)、FAK(Tyr407)、cdc42、RhoC)蛋白表現量關聯性。(四)收集21位肝癌病患在Thalidomide不同治療時期的血清檢體與50位健康人血清檢體，以偵測病患和健康人表達ENO1抗體之程度，其中21位病患加測AFP作為腫瘤變化之指標，以了解anti-ENO1 Ab變化與腫瘤變化二者關聯性。
實驗結果：(一) ENO1 knockdown之B16F1L5 shENO1 2E11 migration程度明顯比對照細胞株B16F1L5 pSM2 vector control低，顯示knockdown ENO1表達會顯著影響腫瘤細胞migration程度。(二)低轉移B16F1細胞株經老鼠體內篩選成高轉移之B16F1L5細胞株後，增加了ENO1 expression；而shRNA knockdown ENO1 expression，致B16F1L5 shENO1 2E11較對照細胞株B16F1L5 pSM2 vector control減少ENO1 expression。ENO1 knockdown之B16F1L5 shENO1 2E11相較於對照細胞株B16F1L5 pSM2 vector control，cdc42、RhoC mRNA的表現因knockdown ENO1 expression而減少。(三) ENO1 knockdown之 B16F1L5 shENO1 2E11相較於對照細胞株B16F1L5 pSM2 vector control，c-Met、FAK、FAK(pY397)、FAK(Tyr407)、cdc42、RhoC蛋白表現量皆因knockdown ENO1 expression而減少。(四) anti-ENO1 Ab變化：健康人主要分布在1:100000以下，病患主要分布在1:150000∼1:250000間，顯示病患有顯著anti-ENO1，其中僅8位病患有明顯AFP值、ENO1抗體關聯性，其餘病患未有明確關聯性。
結論：knockdown ENO1的基因表現，明顯降低migration程度，影響細胞遷移。ENO1 expression可能藉由調控相關訊息而調控腫瘤細胞之migration。依AFP與anti-ENO1之結果未有明確關聯性，可能是檢體數量不夠以致沒有很明顯的趨勢指標。

關鍵字

轉移

並列摘要

It has been reported that the expression level of α-Enolase (ENO1) correlats with the metastasis and survival of patients with lung and liver cancers. The expression of ENO1 may cause invasion and metastasis of tumor cell through many mechanisms. In this study, the correlation between the expression of ENO1 and the proteins related to tumor metastasis was investigated.
Aims of the research:
Part 1: the melanoma cell lines were used to study the relationship between the expression of ENO1 and tumor migration, and to study whether down-regulation of ENO1 expression will influence expression of proteins or genes involved in tumor migration.
Part 2: the changes of immunity against ENO1 in liver cancer patients after thalidomide treatment were investingated.
Materials and Procedures:
The highly metastation melanoma cell B16F1L5 (established after 5 rounds of in vivo selection), B16F1L5 shENO1 2E11(transfected with a shRNA against ENO1), B16F1L5 pSM2 vector control were used in this study. We studied the connection of expression of ENO1 and the migration ability of the tumor cells. Our projects are as following: (1), an in vitro slide wound-healing assay was used to study whether reducing the expression of ENO1 will influence migration of tumor cells or not, (2), RT-PCR assay was used to study whether reducing the expression of ENO1 will influence the mRNA expression of several meatstasis-related genes(uPA, uPAR, MMP-2, MMP-9, cdc42, RhoC) or not, (3), western blotting analysis was used to investigate the relationship between expression of ENO1 and involved in metastasis protein (MMP-2, MMP-9, c-Met, FAK, FAK(pY397), FAK(Tyr407), cdc42, RhoC), (4), ELISA was performed to determine anti-ENO1 antibody level in 21 HCC patients and 50 healthy individuals, and study the changes of anti-ENO1 antibody level in HCC patients after thalidomide treatment.
Result:
Our experimental results were summarized below, First, Down-regulation of ENO1 expression with shRNA will decrease migration of tumor cells, Second, were weakly metastation B16F1 cells were selected to become highly metastation B16F1L5 cells the expression of ENO1 was increased; Down-regulation of ENO1 expression by shRNA reduced the expression of cdc42 and RhoC mRNA, and protein expression of c-Met, FAK, FAK(pY397), FAK(Tyr407), cdc42 and RhoC protein. Fourth, the levels of anti-ENO1 antibody in healthy individuals are lower than those of HCC patients. Only 8 patients have an obvious correlation of AFP value and anti-ENO1 antibody.
Conclusion:
Down-regulation of ENO1 expression decrease the level of migration in B16F1 melanoma cell lines. ENO1 may regulate the migration of tumor cells by several regulatory signals. We can not draw a clear conclusion regarding the levels of anti-ENO1 Ab in patients after thalidomide treatment due to low numbers of patients studied.

並列關鍵字

ENO1 ； metastasis

參考文獻

1. Gee-Chen Chang, Ko-Jiunn Liu, Chia-Ling Hsieh, Tsai-Shu Hu, Suparat Charoenfuprasert, Hsiung-Kun Liu, Kwen-Tay Luh, Li-Han Hsu, Chew-WenWu, Chou-ChikTing, Chih-Yi Chen, Kun-Chieh Chen,Tsung-YingYang,Teh-Ying Chou, Wen-HuaWang, Jacqueline Whang-Peng, and Neng-Yao Shih. Identification of α-Enolase as an Autoantigen in Lung Cancer: Its overexpression Is Associated with Clinical Outcomes. Clin Cancer Res., 12(19): 5746-5754, 2006.

2. Motonari Takashima, Yasuhiro Kuramitsu, Yuichiro Yokoyama, Norio Iizuka, Masanori Fujimoto, Takashi Nishisaka, Kiwamu Okita, Masaaki Oka1 and Kazuyuki Nakamura. Overexpression of alpha enolase in hepatitis C virus-related hepatocellular carcinoma: Association with tumor progression as determined by proteomic analysis. Proteomics, 5:1686-1692, 2005.

3. Lance A. Liotta and Elise C. Kohn. The microenvironment of the tumour–host interface. Nature, 411: 375-379, 2001.

4. Benjamin Terrier, Nicolas Degand, Philippe Guilpain, Amélie Servettaz, Loïc Guillevin, Luc Mouthon. Alpha-enolase: A target of antibodies in infectious and autoimmune diseases. Autoimmunity Reviews, 6:176–182, 2007.

5. Heron Yu and Richard M. Schultz. Relationship between Secreted Urokinase Plasminogen Activator Activity and Metastatic Potential in Murine B16 Cells Transfected with Human Urokinase Sense and Antisense Genes. Cancer research 50: 7623-7633, 1990.