微型核醣核酸34c-5p藉由反向調控AREG-EGFR-ERK路徑可以抑制amphiregulin誘發之卵巢癌幹細胞特性及抗藥性

miRNA-34c-5p inhibits amphiregulin-induced ovarian cancer stemness and drug resistance via downregulation of the AREG-EGFR-ERK pathway

董筱玲(Shiao-Lin Tung)

指導教授 ： 王陸海

國立清華大學/生命科學院/分子醫學研究所/博士(2017年)

摘要

卵巢癌是婦科癌症中最致命的癌症，主要致命原因是因為晚期診斷、容易擴散、以及快速產生抗藥性。癌症幹細胞被認為是產生抗藥性、疾病轉移及復發的重要原因之一。為了探討卵巢癌幹細胞的特性，我們從兩株卵巢癌細胞株SKOV-I6及OVS1成功的培養出類卵巢癌幹細胞。其中OVS1細胞株是直接從卵巢癌病人身上取得的腫瘤培養而成。這些類卵巢癌幹細胞具有重要的癌症幹細胞特性，包括球體形成、自我更新的能力、表現重要的卵巢癌幹細胞標記及表皮間質轉換標記、對藥物的抗藥性增加、以及強力的致瘤性能力。
以基因微陣列分析發現從OVS1細胞株衍生的球形細胞和OVS1原型細胞相較，amphiregulin (AREG) 的表現量上升，而AREG之保守調控的微型核醣核酸34c-5p 的表現量則是下降。以定量即時聚合酶鏈鎖反應分析也證實在兩株細胞株的球形細胞中，AREG的表現量增加以及微型核醣核酸34c-5p的表現量下降。報導基因冷光活性試驗及其突變體分析證實AREG 是微型核醣核酸34c-5p的直接標的基因。實驗分析也發現，AREG調控的球體形成增加、對docetaxel及carboplatin兩種化學治療藥物的抗藥性增加、以及致瘤性能力的增加可以被微型核醣核酸34c-5p所抑制。我們更進一步發現微型核醣核酸34c-5p是透過反向抑制AREG-EGFR-ERK路徑來抑制這些卵巢癌幹細胞特性及抗藥性。實驗也發現AREG的過度表現和卵巢癌病人的晚期分期及不良預後相關。
綜合而論，我們的研究指出AREG藉由AREG-EGFR-ERK路徑來促進卵巢癌幹細胞特性及抗藥性，而微型核醣核酸34c-5p則可反向抑制此路徑。因此藉著抑制AREG，微型核醣核酸34c-5p可以在卵巢癌的抗癌症幹細胞治療中成為非常有潛力的治療策略。

關鍵字

微型核醣核酸34c-5p ； amphiregulin ； 類卵巢癌幹細胞 ； 癌症幹細胞特性 ； 抗藥性 ； AREG-EGFR-ERK路徑

並列摘要

Epithelial ovarian cancer is the most lethal gynecological cancer mainly due to late diagnosis,easy spreading, and rapid development of chemoresistance. Cancer stem cells are considered to be one of the main mechanisms for chemoresistance, as well as metastasis and recurrent disease. To explore the stemness characteristics of ovarian cancer stem cells, we successfully enriched ovarian cancer stem-like cells from an established ovarian cancer cell line (SKOV-I6) and a fresh ovarian tumor derived cell line (OVS1). These ovarian cancer stem-like cells possess important cancer stemness characteristics including sphere-forming and self-renewing abilities, expressing important ovarian cancer stem cell and epithelial-mesenchymal transition markers, as well as increased drug resistance and potent tumorigenicity. Microarray analysis of OVS1-derived sphere cells revealed increased expression of amphiregulin (AREG) and decreased expression of its conserved regulatory microRNA, miR-34c-5p, when compared with the OVS1 parental cells. Overexpression of AREG and decreased miR-34c-5p expression in SKOV-I6 and OVS1 sphere cells were confirmed by quantitative real-time polymerase chain reaction analysis. Luciferase reporter assay and mutant analysis confirmed that AREG is a direct target of miR-34c-5p. Furthermore, AREG-mediated increase of sphere formation, drug resistance toward docetaxel and carboplatin, as well as tumorigenicity of SKOV-I6 and OVS1 cells could be abrogated by miR-34c-5p. We further demonstrated that miR-34c-5p inhibited ovarian cancer stemness and drug resistance through downregulation of the AREG-EGFR-ERK pathway. Overexpression of AREG was found to be correlated with advanced ovarian cancer stages and poor prognosis. Taken together, our data suggest that AREG promotes ovarian cancer stemness and drug resistance via the AREG-EGFR-ERK pathway and this is inhibited
by miR-34c-5p. Targeting AREG, miR-34c-5p could be a potential strategy for anti-cancer stem cell therapy in ovarian cancer.

並列關鍵字

miR-34c-5p ； amphiregulin ； ovarian cancer stem-like cells ； cancer stemness ； drug resistance ； AREG-EGFR-ERK pathway

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