來自於pri-mir-218-1的miR-218可藉由調控N-Cadherin來抑制肺腺癌細胞的移動

MiR-218, Expressed From Pri-mir-218-1, Inhibits Migration of Lung Adenocarcinoma Cells by Suppressing N-Cadherin

王麗茹(Li-Ju Wang)

指導教授 ： 賴亮全

共同指導教授 ： 佘玉萍(Yuh-Pyng Sher)

國立臺灣大學/醫學院/生理學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.02264

摘要

肺癌為造成癌症死亡中最主要的癌症之一，而大腦為肺癌細胞主要轉移的器官。目前臨床上，主要治療腦轉移的方式為放射性治療，但由於放射性治療有很嚴重的副作用，找尋新的且有效率的腦轉移治療方式已成為當前研究中備受關注的議題。目前眾多的實驗治療方法之一，為使用微小 RNA (MicroRNA)去抑制具有腦轉移能力的肺癌細胞，但此領域的發展與對分子層次調控機制的所知相當有限。所以本篇研究中我們將尋找可以抑制肺腺癌細胞轉移的miRNA，並對其分子調控進行探討。
在本篇研究中，我們將使用一具有腦轉移能力的肺癌細胞株─ BM#7 ─作為本篇實驗的研究對象。由於CDH2的過度表現已被證實為細胞轉移的指標，我們利用Illumina miRNA微陣列晶片(microarray)去篩選BM#7細胞與 F4細胞(不具有腦轉移能力的肺癌細胞)相比表現量有差異的miRNA。並利用生物資訊工具去預測並挑選這一群有差異的miRNA中與CDH2之間有鍵結關係的miRNA，最後我們挑選出九個miRNA。由於CDH2在BM#7中表現量上升，我們藉由各種不同的預測方式挑選出與CDH2親和力最高且與CDH2有相反表現的miRNA─ miR-218。我們進一步設計實驗驗證：miR-218的異常表現上升，會促使CDH2表現量下降並抑制肺癌細胞的轉移。
我們先利用即時定量聚合酶連鎖反應 (real-time PCR)去驗證內生性CDH2和miR-218的表現量。結果顯示，在BM#7中miR-218的表現量是下降的，為了瞭解究竟是何種機制促使miR-218下降，因此我們去偵測miR-218的前驅物。由於miR-218的前驅物有兩個分別存在於兩個基因:SLIT2 (pri-mir-218-1)和SLIT3 (pri-mir-218-2)。根據real-time PCR結果表示，miR-218的表現量下降是因為pri-mir-218-1所導致的。之後我們想要再去驗證miR-218是否會與CDH2-3’UTR鍵結，因此我們利用冷光酵素活性測定(Luciferase assay)來證明。結果顯示，miR-218在CDH2-3’UTR的有兩個鍵結位置。最後我們利用細胞穿透試驗 (transwell assay)來探討BM#7細胞中大量表現miR-218在CDH2上的功能影響。由結果看來，大量表現miR-218會抑制BM#7細胞的轉移能力。
綜合以上所述，本篇研究顯示在BM#7中miR-218表現量下降是因為pri-mir-218-1表現量降低所致。此外也證明miR-218可以藉由與CDH2-3'UTR鍵結抑制CDH2表現，進而使細胞轉移能力下降。故miR-218和SLIT2在肺癌細胞腦轉移的臨床治療中，可能可以做為一個新的治療方式與指標。

關鍵字

微小RNA ； miR-218 ； 肺癌 ； 腦轉移 ； SLIT2

並列摘要

Lung cancer is the leading cause of cancer-related mortality in the world. Brain is a major migratory site of lung cancer[1]. So far, the major therapy of brain metastasis is irradiation, but it has a serious side effect. Hence, there is a great need to find efficiency methods for curing brain metastasis. One possible therapeutic strategy is using miRNA to inhibit brain metastatic lung cancer. However, the knowledge of its regulatory mechanism is still very limited. Therefore, as an initial step, the purpose of this study is trying to identify miRNAs that can inhibit the migration of lung adenocarcinoma cells.
Here, a brain metastatic lung adenocarcinoma cell line, BM#7, was used as an experimental model. The expression of CDH2 was used as a biomarker for representing metastatic ability. Illumina miRNA microarrays were used to screen the differentially expressed miRNAs in BM#7 cells and parental F4 cells. Bioinformatic tools were used to predict which miRNA can target to CDH2. We identified nine miRNAs that differentially expressed in BM#7 and predicted to target to CDH2. Among these nine miRNAs, since CDH2 was up-regulated in BM#7, we focused on miR-218 that was down-regulated and predicted to target CDH2 by several algorithms.
The endogenous expression levels of CDH2 and miR-218 were validated by real-time PCR. Next, to understand the regulatory mechanism of miR-218, we examined the expression levels of miR-218 precursors. Since miR-218 had two precursors, pri-mir-218-1 and pri-mir-218-2, located in SLIT2 and SLIT3 respectively, real-time PCR was used to measure their expression levels. The results showed that decline of miR-218 in BM#7 were due to down-regulation of pri-mir-218-1. Third, to explore whether miR-218 could bind to CDH2, luciferase assays were conducted. The results showed that miR-218 could directly bind to CDH2-3’UTR at two binding sites. Lastly, to investigate the function role of miR-218 on CDH2, transwell migration assay was conducted in BM#7 with miR-218 over-expression. The ability of cell migration was suppressed when miR-218 was up-regulated in BM#7 cells.
Taken together, this study showed that the down-regulation of miR-218 was attributed to the decreased expression of pri-mir-218-1 in BM#7, and that miR-218 could inhibit cell migration by targeting to CDH2-3’UTR. MiR-218 might be a novel drug for developing clinical therapies for lung cancer with brain metastasis.

並列關鍵字

MicroRNA ； miR-218 ； SLIT2 ； metastasis ； lung adenocarcinoma

參考文獻

1. Nathoo, N., et al., Pathobiology of brain metastases. J Clin Pathol, 2005. 58(3): p. 237-42.

2. Hoffman PC, M.A., Vokes EE., Lung cancer. Lancet, 2000: p. 479-85.

4. Thun, M.J., et al., Lung cancer occurrence in never-smokers: an analysis of 13 cohorts and 22 cancer registry studies. PLoS Med, 2008. 5(9): p. e185.

6. Grinberg-Rashi, H., et al., The expression of three genes in primary non-small cell lung cancer is associated with metastatic spread to the brain. Clin Cancer Res, 2009. 15(5): p. 1755-61.

7. Hayama, M., et al., One-step Nucleic Acid Amplification for Detection of Lymph Node Metastasis in Lung Cancer. Ann Thorac Cardiovasc Surg, 2013.