探討與食道鱗狀細胞癌相關之miRNAs在細胞增生、爬行、以及抗藥性所扮演的角色

To study the role of recurrence-related miRNAs on cell proliferation, migration, and drug resistance in esophageal squamous cell carcinoma

林玟君(Wen-Chun Lin)

指導教授 ： 蔡孟勳

國立臺灣大學/生命科學院/基因體與系統生物學學位學程/博士(2019年)

https://doi.org/10.6342/NTU201902859

摘要

食道癌東亞地區的男性中致死率排名第五的癌症。由於五年存活率低，許多團 隊都致力於發展更好的治療方法。雖然目前 CCRT 合併手術是最常用的方法，然 而能有效完全去除腫瘤的比例仍然相對較低，而對 CCRT 效果較差的病患則有較 高的復發率與較低的存活率。復發是造成癌症病患的主要原因之一，因此藉由了解 其中的基因調控能有助於癌症的治療。miRNA 在細胞中是重要的調控分子，並且 有研究指出能調控與復發相關的訊息傳遞路徑。因此在這個研究中，利用次世代定 序方式來分析在這群病患中，有復發以及沒有復發的病患的 miRNA 表現量。研究 中，我發現到 15 個有顯著差異的 miRNA，並且藉由細胞實驗方式驗證這些 miRNA 在食道癌細胞中扮演的功能。其中，miR-338-5p 是在復發的病患檢體中表現量較 低的 miRNA，並且在細胞株的分析中，表現量在癌細胞也比較低。接著，我進一 步利用 MTT assay、clonogenic assay、transwell assay 以及 cisplatin 的處理來觀察 miR-338-5p 對於細胞的生長、存活、爬行以及對抗癌藥物的敏感性的影響。此外 我也藉由預測軟體和生物晶片來篩選可能被 miR-338-5p 調控的基因。兩種方法所 篩出的 31 個基因中，FERMT2 在兩個食道癌的大數據中，都能看到其表現與 miR- 338-5p 呈現負相關，此外在 qPCR 與西方點墨法中，能看到 FERMT2 表現量在轉 染 miR-338-5p 的細胞株中有下降，因此進一步以 luciferase assay 研究，並發現 miR- 338-5p 能藉由直接結合 FERMT2 3’ UTR 來調控 FERMT2 的表現。為了瞭解 FERMT2 在細胞增生、爬行、以及抗藥能力中所扮演的角色，除了用 shRNA 降低 FERMT2 的表現外，也在轉染 miR-338-5p 的細胞株中將 FERMT2 大量表現，結果 可發現 FERMT2 能回復一些 miR-338-5p 造成的效應。路徑分析中也能發現，miR- 338-5p 可能藉由 FERMT2 調控 ILK 路徑來影響細胞增生、爬行、以及抗藥性。此 研究能幫助我們了解更多與食道癌復發相關的機制，並對於未來癌症治療方法的 發展有所貢獻。

關鍵字

微小核醣核酸 ； miR-338-5p ； 食道鱗狀細胞癌 ； 癌症復發 ； FERMT2 ； ILK訊息傳遞路徑

並列摘要

Esophageal cancer (EC) is the 5th of leading causes of cancer death among the male population in Eastern Asia. Due to the poor overall 5-year survival rate, effective treatment strategies are needed to improve the long-term survival rate. Although concurrent chemoradiotherapy (CCRT) is the most effective treatment, patients without complete remission still suffer from high recurrence and low survival rate. miRNAs are important gene regulators which also involved in recurrence related pathways. Therefore, I applied Next generation sequencing method to evaluate miRNA expression in recurrence and non-recurrence ESCC patients post CCRT. Fifteen differential expressed miRNAs were identified. Among these miRNAs, miR-338-5p was not only down- regulated in tumor tissue of recurrence patients, but had lower expression level in ESCC cells than normal cells. To understand functions of miR-338-5p, I transfected miR-338- 5p mimic into the ESCC cells (CE-81T), and the results showed that miR-338-5p mimic reduced cell proliferation, survival, migration, and enhanced drug toxicity. Then miR- 338-5p potential target genes were identified utilizing microarray and prediction tools. There were 31 genes with 1.5-fold down-regulation in the miR-338-5p mimic transfected cells. Among these genes, FERMT2 was chosen for further study because it involved in integrin-linked protein kinase (ILK) signaling pathway that was significantly affected by miR-338-5p mimic transfection. In addition, to silence FERMT2 was similar to miR-338- 5p mimic transfection decreasing cell proliferation, survival, migration, and drug resistance. Restoration experiments showed overexpressed FERMT2 rescued the effect of miR-338-5p. This thesis demonstrates that miR-338-5p expression may play a critical role in preventing ESCC recurrence through repressing ILK pathway. My finding proposed that miR-338-5p may be a novel diagnosis marker for the estimation of ESCC recurrence risk, as well as a potential therapeutic component for ESCC treatment.

並列關鍵字

microRNA ； miR-338-5p ； esophageal squamous cell carcinoma ； cancer recurrence ； FERMT2 ； Integrin-linked kinase signaling

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