利用有效之shRNA來研究CRMP-1基因在癌細胞轉移的角色

Role of CRMP-1 in cancer cell metastasis determined by using effective shRNA

江易霖(Yi-Lin Chiang)

指導教授 ： 張富雄

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.2004.10313

摘要

癌細胞的轉移擴散常常造成癌症病人全身性的腫瘤，造成高致死率。研究與癌細胞轉移相關的基因，可以找出對付癌細胞轉移的策略。臺大醫院內科楊泮池教授篩選出轉移能力不同的肺癌細胞 (CL1-0及CL1-5)發現CL1-0細胞的collapsin response mediator protein-1 (CRMP-1) 基因功能正常，所以轉移能力很低；而CL1-5細胞的CRMP-1基因缺陷造成癌細胞轉移能力增加。利用小片段干擾型RNA (shRNA)的方法抑制CRMP-1與其上游Aurora A kinase，可以研究基因的訊息傳遞是如何造成病人的癌細胞擴散。但現今的基因轉殖技術無法兼具高效率、快速、低毒性以及低成本的各項優點，使得運用基因轉殖技術運送質體進到細胞表達shRNA的過程受到許多限制。
本實驗室最近發表了兼具各項優點的基因轉殖技術，稱為”界面基因轉殖” (Surfection)，應用在293T與HeLa細胞可以改進基因轉殖效率分別高達90%與80%，並且可同時有效率的送入三種基因到同一個細胞，並使細胞維持正常的型態。而與傳統的陽離子脂質或聚合物轉殖報導基因luciferase比較，其效力達10倍以上，可篩選具有抑制功能的shRNA。以”界面基因轉殖”技術同時轉殖報導基因與shRNA載體時，其比例只要1:1即可有效的篩選具有抑制功能的shRNA。
有研究指出部分癌細胞的CRMP-1基因表達量有減少的情形，所以可能是屬於metastasis suppressor genes。為了研究CRMP-1可能的基因功能，可以遞送shRNA來衰減CRMP-1的蛋白質表達。有的研究指出要篩選所設計出的shRNA，大約五個shRNA會有一個有抑制目標基因的效果。而目前本實驗篩選三個pAurora A-shRNA已經發現有一個具有抑制效果。本論文首次篩選10個pCRMP1-shRNA，發現有三個具有抑制效果。已經篩選出的pAurora A-shRNA1與pCRMP1-shRNA22可以有效抑制標的基因 (pAurora A-GFP或pCRMP1-GFP) 達到90%。單獨轉染pCRMP1-shRNA22可以有效抑制細胞內生性CRMP-1蛋白質表達90%以上。同時轉殖pAurora A-shRNA1與pCRMP1-shRNA22抑制293T細胞之CRMP-1與Aurora A蛋白質表現，會明顯造成細胞apoptosis的現象增加至5.1%。
根據Angela等人所發表八項設計siRNA的原則，分析本論文所使用的十三個shRNA序列發現有三個shRNA符合其原則，例如pCRMP1-shRNA17符合七項原則；pCRMP1-shRNA22與pAurora A-shRNA-1皆符合六項原則；而其餘十個shRNA序列分別只符合五項以下的原則。
CRMP-1與Aurora A在細胞增生的過程還有許多未知的訊息傳遞機制，將來會再更進一步研究抑制此兩種基因是否會造成細胞凋亡或其他相關基因表現的增減。

關鍵字

界面基因轉殖

並列摘要

Metastasis of cancer cells often lead to a high mortality rate in cancer patients. As metastasis involves a multistep process, blocking of any one of these steps might eventually prevent its completion. Dr. P. C. Yang on our campus previously established several sublines (CL1-0, CL1-1, CL1-5, and CL1-5-F4) from lung adenocarcinoma cells. Comparing the differential expression genes in poorly metastatic and highly metastatic cancer cells, they found that the expression of collapsin response mediator response protein-1 (CRMP-1) mRNA was negatively associated with cell invasiveness.
To investigate the signal transduction pathways involved in metastasis, a new gene silencing method using small hairpin RNA (shRNA) could be applied to functionally knockdown of Aurora A kinase and that of CRMP-1. However, current transfection techniques cannot fulfill high efficiency, high speed and low cell toxicity simultaneously.
We have published a new transfection technique termed “Surfection”, with most of the fore mentioned advantages. Transfction efficiency was improved up to 90% in several mammalian cell lines. Compared to traditional methods of using cationic liposome or cationic polymer, our platform results in 10 times more efficient than traditional methods by the luciferase assays. The ratio of reporter gene to shRNA vector was 1:1, and was sufficient enough to screen the functional shRNA.
Several reports demonstrated that some cancer cells decrease CRMP-1 gene expression. Thus, CRMP-1 may behave as a metastasis suppressor gene. By transfecting three different pAuroraA- shRNAs and ten pCRMP1-shRNAs with the reporter constructs, we have selected one and three effective shRNAs individually. pAurora A-shRNA1 and pCRMP1-shRNA22 were mostly effective in silencing the cognate gene, either in the reporter gene assays or at protein levels.
Furthermore, transfection of functional pAurora A-shRNA1 and pCRMP1-shRNA22 into 293T cells by surfection could increase apoptosis rate up to 5.1% by flow cytometry assay. The effective shRNAs match 6 out of 8 criteria base on the theoretical principles published by Reynolds et al. [Nat. Biotechnol., 22, 326-330].
Even though functions of Aurora A and CRMP-1 in cell proliferation still need to be resolved. we hope further investigation can elucidate their roles in cancer cell invasion by using the powerful tool in cell arrays.

並列關鍵字

surfection

參考文獻

Ball, L. J., Jarchau, T., Oschkinat, H., and Walter, U. (2002). EVH1 domains: structure, function and interactions. FEBS Lett., 513, 45-52.

Bohula, E. A., Salisbury, A. J., Sohail, M., Playford, M. P., Riedemann, J., Southern, E. M., and Macaulay, V. M. (2003). The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J. Biol. Chem., 278, 15991-15997.

Brummelkamp, T. R., Bernards, R., and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550-553.

Byk, T., Dobransky, T., Cifuentes-Diaz, C., and Sobel, A. (1996). Identification and molecular characterization of Unc-33-like phosphoprotein (Ulip), a putative mammalian homolog of the axonal guidance-associated unc-33 gene product. J. Neurosci., 16, 688-701.

Chang, C. C., Shih, J. Y., Jeng, Y. M., Su, J. L., Lin, B. Z., Chen, S. T., Chau, Y. P., Yang, P. C., and Kuo, M. L. (2004a). Connective tissue growth factor and its role in lung adenocarcinoma invasion and metastasis. J. Natl. Cancer Inst., 96, 364-375.