| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 92 === 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.
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