| Summary: | 碩士 === 國立成功大學 === 分子醫學研究所 === 98 === ENO1 encodes a glycolytic enolase 1, catalyzing the conversion of 2-phospho-D-glycerate to phosphoenolpyruvate. Besides, ENO1 also encodes a nuclear MYC-binding protein 1 (MBP1) presumably via alternative initiation at Met97 from one single transcript. In our previous studies, we found that ENO1 overexpression is not only a poor prognostic factor for oral cancer patient survival but also promotes cellular transformation. ENO1 knockdown reduced the stimulatory effect on cell transformation including cell growth, suggesting a tumor promoting role of ENO1. However, the mechanism responsible for ENO1 knockdown-mediated growth inhibition remains unknown. We first confirmed the effect of ENO1 knockdown on cell proliferation, cell cycle progression and apoptosis using cell proliferation and flow cytometry. We found that ENO1 knockdown significantly reduced cell proliferation via increased G1 arrest and apoptosis. By comparing the gene expression profiles of vector control and ENO1 knockdown CAL-27 oral cancer cells using DNA microarray analysis, the mRNA expression of cyclin-dependent kinase inhibitor, p21, was most induced by ENO1 knockdown. ENO1 knockdown-induced p21 mRNA expression was later confirmed by semi-quantitative RT-PCR. When compared the mRNA with protein expression of p21 in two head and neck cancer lines, CAL27 and KYSE170, we found a differential regulation of p21 at transcriptional or post-transcriptional levels. Western blot analysis of protein lysates isolated from knockdown cells treated with actinomycin D or cycloheximide indicates the induction of p21 by ENO1 knockdown can be modulated at transcriptional, post-transcriptional or both. To study the involvement of p21 in the ENO1-knockdown mediated inhibition of cell cycle progression, cell doubling was used to study the effect of p21 knockdown in ENO1-knockdown cells. Knocking down the expression of p21 attenuates the ability of ENO1-knockdown to proliferate, indicating a positive role of p21 in ENO1 knockdown. The inverse expression of enolase 1 and p21 was also detected in several head and neck cancer cells. To differentiate the effect of both ENO1 gene products, enolase 1 and MBP1 on cell proliferation, we fused different ENO1 mutants, ENO1mut1 (encoding MPB only), ENO1mut2 (encoding enolase 1 only), ENO1mut 3 (encoding enolase 1 only) and MBP1, with yellow fluorescent protein (YFP) using site-directed mutagenesis. Western blot analysis confirmed a differential expression of major enolase 1 and minor MBP1 using native translation initiation codons. Consistent with frequent overexpression of enolase 1 in human cancer, YFP-ENO1 fused constructs expressing predominant enolase 1 significantly increased cell proliferation. Although MBP1 encoded by ENO1 was reported to suppress cell growth, the inability of low-expressing MBP1 to reduce cell growth might explain why ENO1 often behaves as a growth promoting gene rather than suppressing gene. Together, glycolytic ENO1 may have an additional role in holding cell cycle in check. The studies of ENO1 and its downstream targets might potentially be explored as potential therapeutic or prognostic targets for treating head and neck cancer.
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