探討SOX2藉由推動S100A14的表現去抑制泌尿道上皮癌細胞的生長及移動能力

SOX2 Inhibits Growth and Mobility of Urothelial Carcinoma by Promoting Expression of S100A14

徐莞婷(Wan-Ting Hsu)

指導教授 ： 李沁

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2014年)

摘要

SOX2 (sex determining region Y-box 2，簡稱 SOX2) 在發育過程及在成體幹細胞的維持裡扮演重要角色，但在腦、肺、乳腺、膀胱癌中有異常表現的現象。SOX2在癌症中扮演的角色與癌症種類有關，例如在肺部表皮上皮細胞中表現SOX2便足以表現出腺癌 (adenocarcinoma) 的產生，但在胃癌中SOX2表現量降低與預後較差有相關性。
在實驗室先前研究中發現，SOX2在膀胱癌細胞BFTC905中有表現，並且也發現到SOX2具有RNA結合的活性，也能改變E1A (Adenovirus early region 1A，簡稱E1A) 剪接報導因子 (splicing reporter) 的選擇性剪接 (alternative splicing)。在我的研究中，首先研究在膀胱癌細胞 (BFTC905) 中剔除SOX2 (knock-down SOX2) 對細胞生長的情形。雖然統計上的差異不明顯，從細胞週期 (cell cycle) 的分析上得知在G1 phase中的細胞比率有略為降低，而在S phase的比率有些微上升，而從生長速度來看，當BFTC905細胞株knock-down SOX2，生長速率有略微的上升，由於在短期內差異並不十分明顯，因此接著透過長時間的觀察來確定，knock-down SOX2對細胞生長是否能有明顯的變化。因此我們將以建立好的shLuc-RFP、shSOX2-EGFP之穩定細胞株混合培養兩週的時間，再去計算細胞數目，而結果也證實shSOX2-EGFP細胞的比率有增加的現象，顯示出knock-down SOX2會讓泌尿道癌細胞生長速度變快；另外，knock-down SOX2也會讓細胞有較好的移動能力，因此在泌尿道上皮細胞癌中SOX2可能做為抑癌基因 (tumor suppressor gene) 之作用。
為了發現被SOX2所調控基因，我們做了大量平行定序的實驗，在knock-down SOX2的BFTC905細胞中，發現S100A14 RNA的表現量降低，而在之前利用共價鍵結免疫沉澱 (Cross-linking before immunoprecipitation，簡稱CLIP) 證實S100A14的RNA會與SOX2進行結合，並且發現SOX2會結合在S100A14的3’UTR上。為了確認SOX2是結合在3’UTR的哪一段區域，我們便製做了一系列S100A14 3’UTR reporter，並觀察SOX2是否會與這些片段進行結合，而結果確實 SOX2會結合在nt643到nt784的3’UTR上片段，因此我的實驗結果提供證據，證實了SOX2會結合在S100A14 mRNA 3’UTR上並進而加強 S100A14的表現。

並列摘要

Sex-determining region Y (SRY)-box protein 2 (SOX2) plays a critical role in stem cell maintenance. However, aberrant deregulation of SOX2 expression is observed in brain, lung, breast, gastric, and bladder cancers. The role of SOX2 in carcinogenesis, however, depends on the types of cancers. While expression of SOX2 in lung epithelium is sufficient for development of adenocarcinoma, down-regulation of SOX2 is correlated with poor prognosis in gastric-type gastric cancer. Our previous study showed that SOX2 is up-regulated in urothelial carcinoma cell line BFTC905. Furthermore, SOX2 exhibits RNA binding activity and is able to modulate alternative splicing of the E1A reporter. Here, we first examined the effect of SOX2 down-regulation to BFTC905 under normal growth condition. Cell cycle analysis indicates a slight decrease to the cells in the G1 phase and simultaneously an increase to the cells in the S phase. Although the difference is too small to establish statistical significance, SOX2 knocked-down BFTC905 indeed displays a slightly faster growth rate. To determine whether knock-down of SOX2 elicits long-term growth advantage, we continuously cultured a mix of BFTC905/shLuc-RFP and BFTC905/shSOX2-GFP for two weeks. The results show that there is a moderate increase to the percentage of BFTC905/shSOX2-GFP in the mixed population, indicating that loss of SOX2 indeed promotes growth competition. In addition to an advantage in growth, BFTC905/shSOX2 also demonstrates higher mobility in both wound healing and the trans-well mobility assays. Thus, in urothelial carcinoma, SOX2 likely functions as a tumor suppressor gene. To identify SOX2-regulated genes, massive parallel sequencing was performed to identify deregulated genes in BFTC905/shSOX2. S100A14, which we previously identified as an in vivo RNA target of SOX2, is also among the genes down-regulated by knock down of SOX2. To characterize how SOX2 modulates S100A14 expression through direct interaction with its mRNA, an oligomer-directed RNase H digestion was coupled to the CLIP assay to demonstrate that SOX2 preferentially binds to the 3’-UTR of the S100A14 mRNA. Using EGFP-S100A14 3’UTR fragment reporters, specific regions of the 3’-UTR were demonstrated to contain the SOX2 binding sites. The results, thus, indicate that SOX2enhances S100A14 expression by binding to the 3'-UTR of the S100A14 mRNA. Knock-down of S100A14 in BFTC905 also leads to an increase of the cells in the S phase and higher mobility, suggesting that SOX2 may exert its cellular functions at least partially by promoting S100A14 expression. Together, our experimental evidences indicate that SOX2 functions as a RNA binding protein in urothelial carcinoma to suppress cell growth and mobility.

並列關鍵字

S100A14 ； SOX2

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