miR-338-5p在食道鱗狀癌細胞中藉由標的HRP-3促進失巣凋亡

miR-338-5p promotes anoikis in esophageal squamous cell carcinoma by targeting HRP-3

蘇雨恩(Yu-An Su)

指導教授 ： 蔡孟勳

國立臺灣大學/生物資源暨農學院/生物科技研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902664

摘要

食道癌目前在全球是所有癌症中第七常見並且死亡率排名位居第六，現今較常治療的方法是化學放射療法。然而，能成功透過此方法治療的患者少於40%，更嚴重的是，有些患者經過化學放射治療後可能會促進癌症的復發。在我們實驗室先前的研究中，我們取了食道癌病患有復發及無復發的腫瘤樣本進行microRNA sequencing分析，結果顯示，miR-338-5p在有復發的病患中，和無復發的病患相比，具有顯著性的低表現量。先前研究更進一步證實過量表現miR-338-5p會抑制食道鱗狀癌細胞CE-81T的生長、遷移、菌落形成以及抗藥性。在本篇研究中，我們著重於研究miR-338-5p抑制CE-81T遷移能力的潛在機制。具有遷移能力的癌細胞可脫離胞外基質並且遷移至其他組織或器官形成續發腫瘤 (secondary tumor)。在癌細胞轉移的過程中，抵抗失巢凋亡 (Anoikis resistance) 是一個關鍵且必要的步驟。因此，根據本實驗室先前的研究，我們在本篇研究中推測miR-338-5p會藉由促進CE-81T失巢凋亡的能力來促成轉移能力降低的結果。
本篇研究中，失巢凋亡試驗 (Anoikis assay) 及軟性瓊膠群落生長試驗 (Soft agar assay) 分別被用於檢測miR-338-5p對於CE-81T失巢凋亡及非錨定依賴性生長 (anchorage-independent growth) 的影響。首先，結果顯示過量表現miR-338-5p可促進CE-81T進行失巢凋亡。為了瞭解哪一個基因是miR-338-5p的目標基因且參與在miR-338-5p對失巢凋亡的調控中，我們證實了HRP-3是miR-338-5p的目標基因且在失巢調控中此基因扮演重要的角色。為了更進一步了解miR-338-5p及HRP-3在失巢凋亡中的重要性，我們從CE-81T中篩選了具有抗失巢凋亡能力的細胞CE-81TAR (CE-81T anoikis resistance)。我們發現CE-81TAR相較於CE-81T，具有較好抵抗失巢凋亡的能力及非錨定依賴性生長能力。除此之外，結果顯示過量表現miR-338-5p及下調HRP-3表現量可降低CE-81TAR對於抵抗失巢凋亡及錨定依賴性生長能力。最後，下調HRP-3表現量，結果顯示CE-81T及CE-81TAR的細胞增殖率皆會下降。綜合以上結果，我們指出在食道鱗狀癌細胞CE-81T中，miR-338-5p會透過標的HRP-3來促進CE-81T失巢凋亡的能力，並進而抑制CE-81T遷移能力下降。

關鍵字

食道癌 ； 細胞轉移 ； 失巣凋亡 ； CE-81T ； miR-338-5p ； HRP-3

並列摘要

Esophageal cancer (EC) is the 7th most common cancer and ranks 6th of mortality worldwide among all the other cancer types. Currently, the most common therapeutic strategy is concurrent chemoradiotherapy (CCRT). However, the respond rate of CCRT remains below 40%, and the other 60% patients may fail to this treatment. Worse than that, some patients may trigger disease progression after CCRT treatment. In our previous study, the tumor samples from EC patients with and without recurrence were performed by microRNA sequencing. The results showed that miR-338-5p was significantly down-regulated in the EC patients with recurrence. Moreover, the further study had shown that overexpressed miR-338-5p inhibited cell proliferation, migration, colony formation and drug resistance of esophageal squamous cell carcinoma CE-81T. In this study, we focused on the underlying mechanisms of inhibiting migration ability by miR-338-5p. Metastatic cells will detach from extracellular matrix and migrate to the other organs to form secondary tumor. Anoikis resistance is a critical step and required during metastasis. Based on our previous study, we hypothesized that miR-338-5p may promote anoikis and lead to decreasing metastatic ability of CE-81T.
In this study, anoikis and soft agar assays were performed to examine the effects of miR-338-5p on anoikis resistance in the CE-81T. The result showed that overexpression of miR-338-5p could increase anoikis in the CE-81T. To understand which genes may be the target of miR-338-5p and involved in regulation of anoikis, HRP-3 was validated as a target gene of miR-338-5p and play an important role in anoikis resistance. To further understand the importance of miR-338-5p and HRP-3 in regulation of anoikis, we generated anoikis resistant enriched subline (CE-81TAR) from adherent CE-81T. We found that CE-81TAR had more capacity of anoikis resistance and anchorage-independent growth than CE-81T. Moreover, overexpression of miR-338-5p and knockdown of HRP-3 could decrease anoikis resistance and anchorage-independent growth in CE-81TAR. Finally, knockdown of HRP-3 was shown to inhibit cell proliferation of CE-81T and CE-81TAR. Taken together, we indicated that miR-338-5p promoted anoikis by targeting HRP-3 and led to decreasing of migration in ESCC CE-81T.

並列關鍵字

Esophageal cancer ； Metastasis ； Anoikis resistance ； CE-81T ； miR-338-5p ； HRP-3

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