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標題: | 利用單一細胞追蹤技術探索SLK與ROCK訊息鏈
在癌細胞遷移過程之交互作用 Using single-cell tracing technique to elucidate SLK-ROCK interaction in cancer cell migration |
作者: | Cheng-Yu Fan 范誠祐 |
指導教授: | 蔡丰喬 |
關鍵字: | 細胞遷移,SLK,ROCK訊息傳遞鍊, Cell migration,SLK,ROCK signaling, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 細胞遷移是一個相當基礎且重要的過程,在胚胎發育、組織修復與癌症轉移中都扮演著重要角色。先前的研究找出了不少調控細胞遷移的基因,但這些基因間如何互相作用則仍然不是很清楚,我們因此進行了一個”two-hit” shRNA screen:同時處理shRNA與小分子訊息抑制藥物,並在內皮細胞上使用傷口癒合試驗以探索這些基因在細胞遷移過程中可能的加乘與拮抗作用。從試驗結果我們找到了Ste20-like kinase (SLK)—一個絲氨酸/蘇氨酸蛋白激活酶,SLK在同時knockdown與加上ROCK抑制劑Y27632時顯著地提升了HUVEC細胞的爬行,因此我們推測SLK與ROCK訊息鏈在細胞爬行中具有交互作用。
為了測試SLK-ROCK交互作用是否為一在各種細胞間普遍存在的現象,我們以同樣平台測試了頭頸癌細胞SAS細胞株。與HUVEC結果不同的是:在癌細胞SAS中進行SLK knockdown,會降低爬行速度,暗示著SLK主要不是藉由改變細胞活動力來影響細胞遷移。因此我們進行了單一細胞追蹤法分析傷口癒合試驗中的每一顆獨立的移動細胞,發現SLK knockdown破壞、而SLK over-expression適度地提升了SAS細胞的移動極性。而我們的觀察與先前文獻也指出SLK會累積在細胞移動前緣,因此我們推測:細胞爬行時SLK會累積在移動前緣並且調控細胞極性。 我們更一步地以單一細胞追蹤法探索了SLK與ROCK訊息鏈之間的交互作用。十分有趣地,ROCK抑制反轉了SLK knockdown所造成的細胞活動力降低,支持了兩分子間具有交互作用的想法。另外我們也發現SLK knockdown能夠反轉RhoA knockdown對細胞動力與黏著斑(focal adhesion)的效果、並且ROCK抑制完全地消除了黏著斑的生成,這些結果顯示在細胞遷移中,SLK對RhoA-ROCK訊息軸線具有重大影響力。此外我們發現ROCK抑制顯著地減少了細胞協調性,此過程可能是透過對黏著小帶(adheren junction) 的調控來達成的。因此SLK與ROCK在細胞遷移的交互作用,也有可能是透過不同的遷移機制調控加成所造成的 (SLK控制細胞極性,而ROCK影響了細胞間的協調性)。 總結以上,我們的結果顯示(1) SLK會累積在細胞移動前緣,調控細胞的移動極性,(2) SLK調控了RhoA-ROCK訊息軸線在細胞遷移中的效果,(3)在細胞遷移上SLK與ROCK可能經由不同機制加成進行交互作用(細胞極性與協調性),更進一步的機轉探討仍然在進行中。藉由這些研究我們將能對SLK與ROCK訊息鏈在細胞遷移上的交互作用有更多認識,並可以此發展嶄新的藥理方針來治療細胞遷移相關的疾病。 Cell migration is a fundamental process in embryonic development, tissue repair and cancer metastasis. Previous studies have identified many genes regulating cell migration, but how these genes interact with each other during cell migration remains unclear. We therefore conducted a “two-hit” screen using shRNAs combined with small molecule inhibitors and scratch wound healing assays in endothelial cells, to identify potential synergistic or antagonistic effects among signaling molecules during cell migration. The screen identified Ste20-like kinase (SLK), a serine-threonine kinase. Its knockdown together with ROCK inhibitor Y27632 drastically increased HUVEC migration. We therefore hypothesized that SLK interacts with ROCK signaling during cell migration. To test if the SLK-ROCK interaction is an universal phenomenon across cell types, we tested the head and neck cancer cells (SAS cell line) on the above platform. In contrast to HUVEC cells, knockdown of SLK in cancer cell line SAS decreased its migration speed, suggesting that SLK might not primarily affect cell motility to change cell migration. We thus performed single-cell tracing techniques to analyze traces of individual moving cell in scratch wound healing assays, showing that SLK knockdown disrupted the migration polarity of SAS cells while SLK over-expression moderately enhanced cell polarity. Together with our observation and previous reports that SLK was accumulated at the migrating cell front, we propose that SLK accumulates at the leading edge of the migrating cell to regulate cell polarity. We further explored the interaction between SLK and ROCK using single-cell tracing. Interestingly, ROCK inhibition reversed the decreased motility caused by SLK knockdown, supporting the idea of interaction between these two molecules. We also noticed that SLK knockdown reversed the effect of RhoA knockdown on cell motility and focal adhesion, and that ROCK inhibition totally abolished focal adhesion formation. All above indicate a pivotal role of SLK on the RhoA-ROCK axis during cell migration. Moreover, we found that ROCK inhibition significantly reduced cell coordination, which probably proceed through the regulation of adherens junctions during cell migration. Hence, SLK and ROCK may also interact with each other by regulating different cell migration modules (polarity vs. coordination.) Taken together, our results indicate that (1) SLK accumulates at the leading edge of migrating cells regulating cell migration polarity, that (2) SLK regulates the RhoA-ROCK axis during cell migration, and that (3) SLK may also interact with ROCK via modular collaboration (polarity vs. coordination.) Detailed mechanistic investigation is still under the way. Through these work we will elucidate how SLK interacts with ROCK signaling during cell migration and develop new pharmacological strategies to treat cell migration-related diseases. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72362 |
DOI: | 10.6342/NTU201803690 |
全文授權: | 有償授權 |
顯示於系所單位: | 藥理學科所 |
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