miR-338-5p藉由調控FERMT2進而抑制食道鱗狀癌細胞的增生、遷移與存活率，並增強對化療藥物的敏感度

MicroRNAs (miRNAs)是一段長度約22-24個鹼基對的小分子核糖核酸，會藉由與mRNA的3端不轉譯區 (3’untranslation region, 3’UTR)進行結合去抑制基因的轉譯達到調控基因的目的。近年來，許多文獻證實miRNA與腫瘤的增生、轉移、藥物敏感度以及細胞癌化程度等有高度相關性。另外，也有研究發現miRNA表現量會與食道癌病患的存活時間或癌症復發等預後情形相關，所以miRNA在腫瘤發展與預後扮演相當重要的角色。我們實驗室先前選取4位復發與4位沒復發的食道癌病患檢體並利用次世代定序平台 (next generation sequencing, NGS)發現miR-338-5p在沒復發的檢體中具有較高的表現量，因此推測miR-338-5p可能與腫瘤復發有相關。在過往文獻中提到miR-338-5p對於其他腫瘤如腦瘤、肝癌等癌細胞的轉移以及放射線敏感度有相關性，但是在食道鱗狀細胞癌中，miR-338-5p的調控機制與影響的相關研究相當少。因此本研究目的，旨在透過細胞實驗探討miR-338-5p在食道鱗狀癌細胞中，與復發相關的腫瘤特性如遷移、增生以及對化療藥物的敏感度等的影響。研究結果發現，我們在食道癌細胞株CE81T/VGH中大量轉染miR-338-5p後透過穿膜試驗 (transwell assay)、MTT assay與細胞群落形成試驗(colony formation assay)發現細胞的遷移、增生速度與存活率都顯著下降。接著透過miRSystem與Ingenuity Pathways Analysis等軟體的預測，發現FERMT2是miR-338-5p可能的調控基因之一，並透過luciferase reporter assay確認。我們更進一步使用FERMT2 shRNA抑制FERMT2表現，並且透過穿膜試驗、MTT試驗與細胞群落形成試驗發現遷移性、增生速度與存活率在降低FERMT2表現量的食道癌細胞中有顯著下降。此外，我們也使用食道癌常用化療藥物cisplatin處理大量轉染miR-338-5p或抑制FERMT2的食道癌細胞，發現不論大量轉染miR-338-5p或抑制FERMT2的食道癌細胞處理cisplatin後存活細胞數都有下降，顯示轉染miR-338-5p或抑制FERMT2都會提高食道癌細胞對化療藥物的敏感度。最後我們使用來自線上資料庫National Center for Biotechnology Information (NCBI)的資料，分析FERMT2的表現程度對食道癌病患存活率的影響，結果顯示FERMT2低表現的食道癌病患有較長的存活時間。總結研究結果，miR-338-5p可以透過抑制FERMT2的表現進而降低食道癌細胞的遷移、增生速度和存活率與增加對化療藥物的敏感度，同時也可利用miR-338-5p的表達程度做為食道癌預後的生物指標。

關鍵字

miR-338-5p ； FERMT2 ；食道癌；細胞遷移；增生；存活率； Cisplatin ；復發

並列摘要

MicroRNAs (miRNAs) are endogenous non-coding RNAs of 22-24 nucleotides in length that regulate gene expression by complementary binding to the 3’ untranslated regions (3’UTR) of specific mRNA targets. In recent years, accumulating evidences suggested that dysregulated miRNAs can function either as tumor suppressor or oncogenes to affect progression, proliferation, metastasis and drug sensitivity of tumor cells. In addition, many studies mentioned that miRNAs were associated with patients’ prognosis, overall survival and recurrence and may play important roles in tumor progression and prognosis. Our previous study showed that the miR-338-5p expression level was higher in the non-recurrence esophageal squamous cell carcinoma (ESCC) patient samples than that in the recurrence ESCC patient samples by next generation sequencing (NGS) analysis. According to this result, miR-338-5p was considered a miRNA that may relate to ESCC patients’ recurrence. Therefore, the aim of this study is to study the possible roles of miR-338-5p and its underlying mechanisms in ESCC cells. We first transfected miR-338-5p mimic into CE81T/VGH cells, which is an ESCC cell line with higher migration, proliferation and survival ability than other ESCC cell lines. The results showed that the cell migration, proliferation and survival were significantly reduced in the cells with mimic-miR-338-5p using transwell assay, MTT assay and colony formation assay. In order to better understand regulatory mechanisms of miR-338-5p in ESCC cells, miRSystem and Ingenuity Pathways Analysis were used to predict the target genes of miR-338-5p. FERMT2 was identified and validated as the target genes of miR-338-5p using luciferase reporter assay. Furthermore, the results showed that the cell migration, proliferation and survival were significantly reduced in the cells with lower FERMT2 expression using transwell assay, MTT assay and colony formation assay in ESCC cell line. Next, we detected the cisplatin induced cytotoxicity in the CE81T/VGH cells and the results demonstrated that the cells with miR-338-5p overexpression or reduced FERMT2 expression could increase cisplatin induced cytotoxicity in the CE81T/VGH cells. Moreover, we analyzed the relationships between FERMT2 expression levels and the survival time of ESCC patients, and the result indicated that FERMT2 could be a good biomarker to predict the survival outcome of esophageal cancer patients. In summary, our study suggested that miR-338-5p might reduce cell proliferation, migration, survival, and to enhance the cisplatin sensitivity in the ESCC cells through FERMT2 regulation.

並列關鍵字

miR-338-5p ； FERMT2 ； ESCC ； Migration ； Proliferation ； Survival ； Cisplatin ； Recurrence

參考文獻

5. 陳旺全, 食道癌之中醫治療. 中醫藥研究論叢, 2009. 12(1): p. 45-60.

1. Fitzmaurice, C., et al., The Global Burden of Cancer 2013. JAMA Oncology, 2015. 1(4): p. 505-27.

2. Stewart, B., et al., World cancer report 2014. World Cancer Report, 2015.

3. Zhang, H.-Z., et al., Epidemiologic differences in esophageal cancer between Asian and Western populations. Chinese Journal of Cancer, 2012. 31(6): p. 281.

4. Croghan, G.A. Gastrointestinal oncology: principles and practices. in Mayo Clinic Proceedings. 2002. Gastroenterology.

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miR-338-5p藉由調控FERMT2進而抑制食道鱗狀癌細胞的增生、遷移與存活率，並增強對化療藥物的敏感度

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