| Summary: | 碩士 === 國立中正大學 === 生命科學系暨分子生物研究所暨生物醫學研究所 === 98 === Early epidemiological studies had established that the counties of Southwestern Taiwan with unusually high prevalence of transitional cell carcinoma (TCC) are also those with high incidence frequency of Blackfoot disease. Nevertheless, close inspection of the earlier reports indicated that the populations with high incidence frequency of TCC and of Blackfoot disease are different, implying that in additon to arsenic contamination other previously unidentified molecular mechanism is responsible for occurrence of TCC in this region. Recent advances have indicated that alternative splicing could play a pivotal role in oncogenesis. In an effort to investigate the role of aberrant splicing in development of TCC, we discovered that the splice site utilization in TSGH8301 and BFTC905, cell line derived from the patients in Taiwan, has been altered. Through gene expression profiling, it was found several proteins potentially functioning in RNA metabolism, including SRY-related HMG-box 2 protein (SOX2), were upregulated in the TCC cell lines. In order to determine whether SOX2 participates in splicing directly, we have purified full-length SOX2 protein and HMG-domain-deleted SOX2 for in vitro pull-down assays and found that SOX2 exhibits RNA-binding capability. Through SELEX, we also identified putative consensus SOX2 binding sequence and currently are validating the result of SELEX by EMSA. Moreover, a HeLa cell line stably expressing FLAG-tagged SOX2 was established, and we found the expression level of SOX2 protein in G2 phase were significantly higher than G1 phase. Hence, we initiated the investigation into the post-transcription mechanisms that regulate expression of SOX2. To examine this possibility, a reporter plasmid with 3’UTR of SOX2 inserted to the downstream of EGFP ORF has been constructed and used to evaluate whether expression of SOX2 protein is also regulated at the translation level. Simultaneously, we also profiled the miRNA expression in TCC by Human miRNA microarray. The expression level of SOX2 in several TCC specimens was also examined by immunohistochemistry, and the preliminary result indicates upregulation of SOX2 in all TCC samples. Additional specimens will be obtained and evaluated upon approval of IRB, Buddhist Dalin Tzu Chi General Hospital. Through this study, we hope to confirm the potential role of SOX2 in pre-mRNA splicing, adding SOX2 to the growing lists of transcription factors that also play the functions of splicing factors.
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