TWI473880B - 微型核糖核酸作為癌症進展之預測因子及其治療癌症之用途 - Google Patents
微型核糖核酸作為癌症進展之預測因子及其治療癌症之用途 Download PDFInfo
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Description
本發明係關於微型核糖核酸(microRNA)作為癌症進展之預測因子及其治療癌症之用途。
多形性神經膠質母細胞瘤(Glioblastoma multiforme，GBM)、第IV級神經膠質瘤為最具惡性的主要腦部腫瘤，且具有相當不良的預後(Stupp et al.,2005)；儘管結合多種療法，其存活中位年數仍約為12個月(Dehdashti et al.,2006;Stupp et al.,2007;Stupp et al.,2005)。為了提高病患之存活，故需瞭解GBM的腫瘤形成機制。部分研究指出癌幹細胞(cancer stem cell，CSC)的亞細胞群係為一主要的因素使癌細胞具抗放射線性，且亦與腫瘤發展，及經傳統療法後之癌症週期性復發有關係(Bao et al.,2006)。然而，迄今仍無適當的指標(marker)可用以分離癌細胞之該等重要的亞細胞群，根據CSC之腫瘤形成假說，該等亞細胞群於活體內(in vivo
)具有重新形成新腫瘤的能力，且與腫瘤復發為惡性神經膠質瘤有關係(Chen et al.,2010)。
微型核糖核酸(miRNAs)為高度保留的小型RNA分子，其可調控基因表現-可做為癌症之標記、致癌基因或腫瘤抑制劑(Croce,2009)。MiRNAs可能可標靶致癌基因、細胞週期調控子及轉錄因子，且可調控腦部腫瘤之發展(Gillies及Lorimer,2007)。於腦部腫瘤中，多種miRNAs，包括miR7、miR21、miR26a、miR124、miR137、miR184、及miRNA 221/222與GBM致病機轉有關連(Chan et al.,2005;Chen et al.,2008;Diehn et al.,2009;Huse et al.,2009;Kefas et al.,2008;Li et al.,2009;Malzkorn et al.,2009)。多種miRNAs可能可做為預後的指標，像是miR10b及miR26a可於高度的神經膠質瘤作為預後的指標(Huse et al.,2009;Sasayama et al.,2009)。miRNAs涉及腦部腫瘤發展之許多方面，包括神經膠質瘤惡化發展。miR125b、miR326、及miR324-5p可於小腦神經性前驅物及腫瘤中作為標記型miRNAs，其有助於預測預後及病患病況(Ferretti et al.,2008)。再者，過量表現miR34可降低腦部腫瘤及胰臟癌幹細胞(CSCs)的自我新生能力(Ji et al.,2009b)。近來，miR145被發現可調控胚胎幹細胞之分化；該單一的miRNA同時地可調控多種幹細胞性基因，包括KLF4、Oct4、及Sox2(Xu et al.,2009)。然而，目前對於GBM之復發，以及藉由調控幹細胞性(stemness)以調控二次的GBM復發、腫瘤誘發能力、或間葉轉化，miRNAs是否於其中扮演該等角色皆尚未明瞭。
miR142為第一個被報告具有可用以調控造血作用及T細胞發育之能力(Chen et al.,2004)。miR142-3p的表現係藉由LMO2結合至miR142基因之推測的啟動子區域而調控(Yuan et al.,2008)。該miR142基因位於t(8;17)易位之交界處，其可能與惰性淋巴瘤發展為急性的B細胞淋巴瘤有關，此可能歸因於c-Myc高度地向上調控(Gauwerky et al.,1989)。Qian等人(2008)發現於老鼠肺臟之支氣管肺泡幹細胞(bronchioalveolar stem cell，BASCs)的miR142-3p及miR142-5p為向上調控的；異常的miR142表現可能與將BASCs轉變為肺癌幹細胞有關。近來，Sun等人(Sun et al.,2010)指出miR-142可用以弱化造血細胞之增生作用，係藉由miR-223-CEBP-β-LMO2軸調控。分離自神經膠質瘤或GBM之CSCs具有提高表現的幹細胞性基因，該基因像是Sox2(Gangemi et al.,2009)。Sox2為高移動性群組之DNA結合蛋白，為神經幹細胞之重要指標，且對於維持該神經幹細胞於未分化狀態十分重要。相同地，Sox2之表現被推測可作為神經膠質瘤及神經管胚細胞瘤，以及維持CSC幹細胞性之指標(Decarvalho et al.;Sutter et al.)。重要的是，神經膠質母細胞瘤幹細胞具有高度的Sox2表現，且將Sox-2剔弱(knockdown)可進一步抑制致腫瘤性及神經膠質瘤CSCs之幹細胞性(Decarvalho et al.)。
本發明係基於發現miR142-3p之新穎功能，其在於miR142-3p可調控Sox2、腺苷酸環化酶9(adenylyl cyclase 9，AC9)、及CD133之表現，以及進而調控復發性的GBM細胞及CSCs細胞之整體幹細胞性，且於復發性GBM細胞中，該miR142-3p可調節其腫瘤誘發特性。本發明進一步提供一種以微型核糖核酸(miRNA)為主之新穎療法，可用以治療腦瘤。
因此，於一方面，本發明提供一種於細胞內抑制目標基因包含CD133、Sox2、及AC9之表現的醫藥組合物，其包括有效量之miR142-3p及醫藥上可接受之載體。
在一具體實施例中，該miR142-3p係直接與目標基因之mRNA的3’端非轉譯區(3’UTR)連結。
在另一具體實施例中，該細胞為一復發性多形性神經膠質母細胞瘤(GBM)細胞。
在另一方面中，本發明提供一種用以診斷或預測個體於治療後，是否具有或具有發展為復發性多形性神經膠質母細胞瘤的風險之方法，包含：
(a) 測量來自患有原發性GBM之個體的對照組樣本中miR142-3p之水平；
(b) 經治療後，測量來自相同個體之試驗組樣本中miR142-3p之水平；以及
(c) 將步驟(b)之試驗組樣本中miR142-3p之水平與步驟(a)之對照組樣本中miR142-3p之水平進行比較，
其中當步驟(b)之試驗組樣本中之miR142-3p之水平低於步驟(a)之對照組樣本中miR142-3p之水平，係代表經治療後，該個體具有或具有發展為復發性多形性神經膠質母細胞瘤的風險。
在一具體實施例中，於上述方法中，該個體已接受過外科手術治療、化學治療、放射性治療、或其組合。
在另一具體實施例中，於上述方法中，該個體為患有第I、II、III、或IV級神經膠質瘤之病患。
於又一方面中，本發明提供一種將miR142-3p用於製造治療多形性神經膠質母細胞瘤之醫藥組合物的用途。該醫藥組合物可藉由立體定位裝置以投與至腫瘤所在位置。該多形性神經膠質母細胞瘤可為復發性多形性神經膠質母細胞瘤。該miR142-3p可為具有經LNA-及硫代磷酸酯-修飾骨架之修飾的微型核糖核酸(microRNA)。
於一具體實施例中，本文所用之miR142-3p係由核酸所編碼。該核酸係位於載體上，其中該載體係選自由質體、黏質體、噬菌質體、及病毒所組成之群組。
於再一方面中，本發明提供一種用以產生多形性神經膠質母細胞瘤動物模式之方法，包含：
(a) 準備原發性神經膠質瘤細胞；
(b)將miR142-3p反義寡核苷酸導入該原發性神經膠質瘤細胞，以得到miR142-3p缺失的細胞；
(c) 將該miR142-3p缺失的細胞植入動物中，以得到患有多形性神經膠質母細胞瘤之動物。
於再一方面中，本發明提供一種將miR142-3p用於製造增加多形性神經膠質母細胞瘤細胞對於放射性療法敏感性之醫藥組合物的用途。
本發明之各種具體實施例係詳細揭露如下。其他本發明之特徵將藉由下述關於各種具體實施例及申請專利範圍的詳細說明及圖式清楚地呈現。
吾人咸信屬於本發明領域之具有通常知識者，可基於本文之敘述，且不需進一步闡述，即可將本發明應用至最寬廣之範圍。因此，應理解的是，以下說明僅為示例性目的，而非以任何方式用於限制本發明之範疇。
本發明將參閱以下具體實施例以更特定地說明，該等具體實施例僅用以示例之目的，而非用以侷限本發明。
神經膠質母細胞瘤(GBM)為最常見之主要的腦部腫瘤，且其預後通常不佳。此外，於多數病患中，經放射化療後會在數月內復發。於本發明中，於復發性GBM之miR142-3p表現量較原發性GBM低，且具有同時表現(co-expression)的Sox2和AC9以及被抑制的miR142-3p之GBM病患會導致較差的預後及存活率。藉由標靶Sox2及AC9，MiR142-3p可調控復發性GBM的腫瘤誘發能力及間葉轉化。於復發性GBM中增加的miR142-3p可降低致腫瘤性、間葉轉化及抗放射線性；當於原發性GBM中miR142-3p為向下調控時，則會促進腫瘤誘發、抗放射線性及間葉轉化，此點可能可用於GBM病患之病症發展及復發的指標。因此，miR142-3p可能涉及CSC重新編程改造(reprogramming)的轉錄網絡，以及涉及於腦部腫瘤中調控間葉轉化。更重要地，LNA-修飾的miR142-3p寡核苷酸可有效地阻斷復發性GBM所衍生的原位異種移植物之腫瘤之生長以及間葉轉化特性，以及可協同增強放射療法之功效。目前就吾人所知，本發明為第一個發現及證明miR142-3p可作為癌症類幹細胞特性(cancer stem-like property)及GBM復發的一重要調控因子，且該miR142-3p可能為一具潛力之臨床預後指標，以及一種新穎的以miRNA為基礎之GBM療法。
對於miRNAs用於致腫瘤性之應用日趨被重視，越來越多之證據指出miRNAs涉及類CSC特性(Garzia et al.,2009;Ji et al.,2009a;Ji et al.,2009b;Silber et al.,2008)。例如，異位表現let-7、miR200c、及miR30可抑制乳房之CSCs的致腫瘤性(Shimono et al.,2009;Yu et al.,2010)。近來，Iliopoulos及其同僚報告miR200b可透過直接標靶Suz12以調控CSC特性，該Suz12為一多齒梳抑制子複合物(polycomb repressor complex)之次單位(Iliopoulos et al.,2010)。於本發明中，miR142-3p可直接標靶Sox2、AC9、及CD133 3'UTRs，以及藉由抑制復發性GBM細胞之Sox2及AC9表現來壓抑其幹細胞性及致腫瘤性。更重要地，將miR142-3p表現剔弱，可明顯地活化GBM-CSC自我新生能力，以及抑制多能性基因的表現。再者，吾人證實SPONGE-miR142-3p可於活體內(in vivo
)增加原發性GBM之腫瘤誘發能力約10至10000倍(圖5H及圖15)。此證據支持了於GBM中向下調控miR142-3p表現時，會導致類幹細胞特性，此即給予GBM細胞誘發及重新生成新腫瘤之能力。再者，已知Sox2於調控神經膠質瘤幹細胞之腫瘤誘發能力上扮演一重要角色(Gangemi RM,. 2009)。過量表現Sox2會促進神經管胚細胞瘤腫瘤誘發細胞生成，以及與神經管胚細胞瘤病患存活率呈現負相關性(Sutter et al.,2010)。資料進一步證實同時剔弱(co-knockdowm)Sox2及AC9，而沒有剔弱CD133，會抑制GBM細胞之腫瘤誘發能力及抗放射線性，此點可藉由miR142-3p之向下調控誘發(圖4、圖7及圖15)。miR142-3p可負調控幹細胞性基因之表現，而降低miR142-3p表現會促進CSC相關的自我新生及抗放射線性。進一步探討miR142-3p所調控之GBM或GBM-CSC之去分化作用(de-differentiation)或重新編程改造，將可更深入瞭解miR142-3p於GBM發展上所扮演的角色。
癌幹細胞(CSCs)已於尤文氏肉瘤(Ewing sarcoma)家族性腫瘤中被發現，其仍保有間葉幹細胞(MSC)的可塑性；EWS-FLI-1及miRNA-145係以相互抑制的回饋循環方式互動、辨識其共同標靶基因Sox2，並接著誘發MSC重新編程改造以轉變成尤文氏肉瘤CSCs(Riggi et al.)。於自我新生之人類胚胎幹細胞中miR145表現為低的，且內源性miR145會抑制多能性因子Oot4、Sox2、及Klf4之3'
UTRs(Xu et al.,2009)。近來，完整的基因體分析顯示異常的基因表現會決定GBM之典型、間葉型及原神經之類型(Verhaak et al.)，且進一步發現神經膠質瘤特異性的調控網絡，其中涉及一轉錄模組，其可活化惡性神經膠質瘤之間葉基因表現(Carro et al.,2010)。因此，miRNAs可能調控腦腫瘤及GBM-CSCs之自我新生及間葉特性。此資料證實，miR142-3p表現與GBM之發展為負相關性，且將原神經型GBM細胞之內源性miR142-3p靜默可誘導該等細胞重新編程改造以轉變為間葉型態或GBM之CSCs。再者，同時表現Sox2及AC9亦可調控GBM之間葉特性。於復發性M型GBM細胞中，同時消除Sox2及AC9則會促進間葉性特徵性改變為原神經性之外表型，而當Sox2及AC9同時過量表現於原發性GBM細胞時，將重新編程改造原神經性之外表型轉變為間葉的特徵性。因此，miR142-3p可能為Sox2及AC9之上游調控因子，以調控間葉及原神經性的轉變。此外，高度GBM之分子指標包括葡萄糖作為主要神經元細胞能量之來源，以及IDH1基因之突變(Zhao et al.,2009)。既然cAMP可調控丙酮酸鹽脫氫酵素(Pyruvate dehydrogenase)，cAMP可能可調控糖解路徑以及改變腦部之葡萄糖依賴性(Huang et al.,2009)。cAMP之生成以及糖解代謝路徑之改變是否與IDH1所調控之TCA循環阻斷相互作用仍屬未知。然而，cAMP及IDH1皆涉及糖解路徑，且cAMP為二級訊息傳遞者。因此，若能發現GBM之AC9受器，將可有助於找到新穎之GBM療法。
CD133，一5-穿膜糖蛋白，為造血幹細胞及內皮前驅物之指標，且可能涉及血管新生作用(Corbeil et al.,2000;Hilbe et al.,2004)。CD133表現已被推測可作為神經膠質瘤之腫瘤再生長、惡性發展、及腫瘤狀況之預後指標(Zeppernick et al.,2008)。近來由Pallini等人所提出之存活率報告(Pallini et al.,2010)，提出相較於原發性GBM之CD133陽性細胞百分比，於復發性GBM之CD133陽性細胞百分比增加4.6倍，而增加CD133之表現與腫瘤復發後之較長的存活率有明顯的相關性。於本研究中，免疫組織化學分析顯示，相較於原發性GBM，復發性GBM之CD133陽性細胞之百分比明顯的增加。然而於存活率分析顯示，CD133表現量與GBM之存活率並無明顯相關性(圖2)。值得注意的是，於原神經型GBM之CD133 cDNA過量表現或於間葉型GBM以siRNA靜默的CD133，於活體外(in vitro
)及活體內(in vivo
)並不會影響間葉的外表型表現或致腫瘤能力。值得注意的是，相較於CD133+
GBM細胞，CD133-
細胞被發現於神經球誘發效率及細胞聚落生成性上具有相似的CSC特性((Beier et al.,2007)；(Joo et al.,2008)；(Ogden et al.,2008)；(Wang et al.,2008))，以及部分PTEN缺乏的GBM腫瘤被發現可產生自我新生之腫瘤誘發CD133+
及CD133-
細胞型態(Chen et al.,2010)。因此，進一步亟需探討以判定CD133表面抗原或CD133相關的路徑是否參與類幹細胞特性之調控，以及CD133是否可做為預測GBM病患存活率之指標或神經膠質瘤CSCs之治療標的。
總結，miR142-3p可調控GBM之腫瘤誘發特性及間葉轉化能力。升高的miR142-3p表現會降低癌症類幹細胞特性及幹細胞性；抑制miR142-3p會增加GBM之致腫瘤特性。因此，miR142-3p可能為治療腦部腫瘤之一種新穎的治療範例。
所有取得組織之程序皆依照赫爾辛基宣言(Declaration of Helsinki)，且經台北榮民總醫院科學研究與倫理審查委員會評估後始進行。簡言之，將GBM組織手術摘除後，以含有HBSS之葡萄糖沖洗該組織三次，接著將樣本以300 mm之厚度進行切片，並將切好的組織浸泡於含有葡萄糖及HBSS之0.1%(w/w)之膠原蛋白酶中，於37℃作用15分鐘，並用搖晃震盪機以125 rpm速度進行震盪培養。將GBM初代培養及GBM細胞株培養於含有10%胎牛血清之MEM(Invitrogen,Carlsbad,CA,USA)中，且於MEM中添加1 mM丙酮酸鈉、非必需胺基酸、2 mM L-麩胺酸、100 units/mL青黴素、及100 μg/mL鏈黴素，並於標準培養環境下進行培養(37℃、95%潮濕空氣、5% CO2
)。
所有取得組織之程序皆依照赫爾辛基宣言，且經台北榮民總醫院科學研究與倫理審查委員會評估後始進行。簡言之，將頭頸部之癌組織以手術摘除，並以含有HBSS之葡萄糖沖洗該組織三次，接著將樣本以300 mm之厚度進行切片，並將切好的組織浸泡於含有葡萄糖及HBSS之0.1%(w/w)之膠原蛋白酶中，於37℃作用15分鐘，並用搖晃震盪機以125 rpm速度進行震盪培養。接著將經膠原蛋白酶消化後之細胞，以100xg之速度進行離心10分鐘，並將該等細胞重新懸浮於癌症幹細胞富集培養基中以培養GBM細胞，其中該富集培養基為添加有無血清DMEM/F12(GIBCO)培養基、N2補充物(R&D)、10 ng/mL人類重組的bFGF(R&D)及10 ng/ml EGF之DMEM/F12培養基。
為了分離CD133陽性細胞，其分離步驟如下所示：將自GBM病患之腫瘤樣本所分離的細胞以1 mL CD133/1之微磁珠/106
細胞進行標記，其係使用CD133細胞分離套組(MACS,Miltenyi Biotec)。將CD133+
細胞培養於添加有N2補充物(R&D)、10 ng/mL人類重組的bFGF(R&D)及10 ng/ml EGF之無血清DMEM/F12(GIBCO)培養基(Chiou et al.,2008;Kao et al.,2009;Kota et al.,2009)。
以Theratronic cobalt unit T-1000(Theratronic International,Inc.，渥太華，加拿大)傳送伽瑪放射線(游離輻射；IR)，劑量為1.1 Gy/min(SSD=57.5 cm)。將對照組之細胞及IR組的細胞暴露在不同劑量下(0、2、4、6、8、及10 Gy)。經10天培養後，將細胞聚落(每個細胞聚落超過50個細胞)固定，並以結晶紫及甲醇溶液進行染色20分鐘。以細胞聚落形成試驗分析細胞存活率。培養效率(Plating efficiency，PE)及存活部分係以下式計算：PE=(細胞聚落數目/接種細胞數目)×100%；SF=計算而得之細胞聚落數目/(接種細胞數目×(PE/100))。
分離腫瘤細胞，並於2×106
活細胞/75 cm2
角瓶中，培養於添加有N2補充物(R&D)、10 ng/mL表皮生長因子(EGF,Invitrogen)、10 ng/mL鹼性纖維母細胞生長因子(bFGF，Invitrogen)、及青黴素/鏈黴素的改良之DMEM/F-12，使成為腫瘤球。經14天後，計算腫瘤球數目。
根據製造商之操作手冊(Invitrogen)，自細胞或組織中以Trizol試劑製備全部的RNA。用於qRT-PCRs之mRNAs以Superscript III第一股合成系統進行反轉錄後，以進行RT-PCR(Invitrogen)。將所得之cDNAs於ABI 7900HT(Applied Biosystems)進行qRT-PCR反應。所用到之引子序列分列於表1中(SEQ ID NOS. 1-24)。
對於miRNAs，使用TaqMan miRNA試驗及特異性引子對以進行qRT-PCR。所有試劑及試驗方法皆來自Applied Biosystems，且以7900HT快速即時PCR系統進行偵測。所使用之引子對皆列於表2(SEQ ID NOS. 1-24)。
所有的質體皆被評估驗證及定序過。SOX2、ADCY9、及CD133 3'UTRs係以PCR增幅，並將之構築於pMIR-RE PORT載體(Applied Biosystems)上。並藉由Sox2(SEQ ID NOS. 25-29)、ADCY9(SEQ ID NOS. 30-35)、及CD133 3'UTRs之引子組(SEQ ID NOS. 36-41)生成各種序列缺失之構築體(表3)。
藉由PCR及具有校正功能之Taq以增幅各種不同的SOX2、ADCY9、及CD133 3'UTR區域。藉由引子對將額外之限制酶切位(MluI/HindIII或SpeI/PmeI)引入；再以MluI/HindIII或SpeI/PmeI將增幅區域酶切後，將之再構築於pMIR-螢光酵素報信子質體上。至於miR142-3p sponge之寡核苷酸，miR142-3p反義及不規則的(scramble)構築已列於表3(SEQ ID NOS.42-52)。如同Gangemi et al.,2009所述方式，將寡核苷酸黏合後將之接合至BLOCK-iT Pol II miR RNAi表現載體上。標靶人類SOX2序列之寡核苷酸序列皆已設計於Invitrogen網站上。選定其中一個寡核苷酸，將之如同Gangemi et al.,2009所述之方式構築入BLOCK-iT Pol II miR RNAi表現載體。構築用引子對(SEQ ID NOS. 42-52)皆列於表3。該pcDNA 6.2-GW/EmGFP-miR-neg對照組質體含有一插入子，其可形成髮夾結構，可修飾成成熟的miRNA，但其被設計為不會標靶任何已知脊椎動物基因。無5’突出端(overhang)之負對照組序列列於表3。所有構築方法皆按照製造商之操作手冊執行。
根據製造商之操作方式進行細胞標記，以含有藻紅蛋白(Miltenyi Biotech.,Auburn,CA,USA)之抗CD133抗體將細胞染色。以FACSCalibur(Becton Dickinson)及CellQuest軟體測量受藍色(488 nm)激發光照射的10,000的細胞之紅色(>650 nm)螢光發射光。於細胞分選試驗中，將細胞標記並以FACSAria(BD Biosciences)進行分選。
以如同miR142-3p之序列合成帶有修飾的miR142-3p RNA寡核苷酸。將硫代磷酸酯骨架修飾為磷酸二酯骨架，且將核糖以鎖核酸(lock-nucleic-acid，LNA)核糖取代之。miR142-3p angomir之運送係藉由微脂體為主的核酸運輸方法所調控。簡言之，將miR142-3p溶解於D5W(5%葡萄糖水)中，並以特定的脂質成分包埋入微脂體中。將包好的微脂體(miR142-3p angomir之最終濃度為10ng/μL)送入顱內異種移植的腫瘤誘發細胞的相同位置。
以miVana miRNA分離套組(Ambion)或Trizol(Invitrogen)將microRNAs萃取出來。將50 ng之分離的小型RNAs，置入每一個孔中進行電泳，其中該電泳係於含有7M尿素之15%聚丙烯醯胺凝膠之0.5X TBE中進行。經電泳後，藉由半乾式轉移機將小型RNAs轉移至Hybond-N+膜上，並藉由UV交聯機進行交聯。以miVana探針及標記套組合成RNA標記(decade marker)，並藉由相同套組將對於miR142-3p特異性之LNA-RNA寡核苷酸(SEQ ID NO.52)(表3
)進行標記，以引入5’端之放射性標記。將膜片先進行前雜交(prehybridized)，再以雜交緩衝液進行雜交處理24小時，再以沖洗緩衝液進行沖洗三次。雜交訊號曝露於X光片48小時。
將細胞於室溫下固定於含4%三聚甲醛之PBS中15分鐘，接著以含有0.1% NP-40及0.1% X-100 Triton之PBS將細胞進行通透化。經以10%驢血清，於室溫封閉(blocking)2小時後，將LNA修飾的，及FITC共軛結合的miR142-3p反義寡核苷酸(SEQ ID NO.52)置入雜交緩衝液中，於42℃作用整夜，接著以5X SSPE及2X SSPE清洗。染色影像係以Olympus影像系統(Silahtaroglu et al.,2007)拍攝。寡核苷酸序列(SEQ ID NO. 52)列於表2。
北方墨點法用以偵測miR142-3p表現
鎖核酸修飾的反義miR142-3p
原位雜交用以偵測miR142-3p表現
鎖核酸修飾的反義miR142-3p，其與FITC共軛結合
以慢病毒為基礎的Sh-RNA的標靶序列
細胞蛋白萃取及免疫墨點法分析係根據(Kao et al.,2009)所述實行。將15μL樣本於95℃煮沸5分鐘，並於10% SDS-PAGE上分離。將該等蛋白藉由濕轉漬方式(wet-transfer)轉漬至Hybond-ECL硝化纖維紙或PVDF膜上(Amersham,Arlington Heights,IL,USA)。添加所述之一級抗體及二級抗體。有反應的蛋白條帶係由ECL偵測系統(Amersham)偵測。所用到之抗體如表3所列。
將細胞再培養於蓋玻片上，以4%三聚甲醛固定，再以0.1% Triton X-100將細胞進行通透化，並以所述抗體進行免疫染色，再以FITC-或PE-標記的二級抗體進行染色影像化。
將病患之組織樣本置於玻片上以進行免疫組織化學染色。經脫蠟及復水後，將1X Trilogy稀釋於H2
O中後(Biogenics)，加熱以使組織切片進行抗原修復。將切片浸泡於3% H2
O2
10分鐘，且以PBS沖洗三次。將組織切片以血清封閉(blocking)(Vestastain Elite ABC套組，Vector Laboratories，Burlingame，CA，USA)30分鐘，接著在室溫下於PBS溶液中將之與一級抗體之抗人CD133及鼠抗人之Sox2(Cell Signaling Technology)培養2小時。以PBS沖洗組織切片，並與生物素標記之二級抗體進行培養30分鐘，接著以標記有卵白素-辣根過氧化酶之結合物進行培養30分鐘，並以PBS沖洗三次。將組織切片浸泡於染料3-3’-二胺基苯啶及H2
O2
受質溶液(DBA/Ni受質套組，SK-4100，Vector Laboratories)中作用10分鐘。使用蘇木精(Hematoxylin)以進行對比染色(Sigma Chemical Co.)。最後，將腫瘤切片以(BDH Laboratory Supplies，UK)固定於蓋玻片上，並以顯微鏡觀察。病理專家在不知臨床資料情況下，將免疫組織化學結果進行計分。經觀察每張切片之五個高解析視野後以進行解讀，且每個視野計算100個細胞供分析。
使用帶有聚碳酸酯濾膜(8-μm孔徑大小；Corning，英國)之24孔盤Transwell系統。將細胞懸浮液種於Transwell之上方反應室，以1×105
細胞密度種於100 μL無血清培養基。將該濾膜的反面，朝向下方反應室，以Hoechst 33342染色3分鐘，接著以倒立式顯微鏡觀察移動中的細胞。為了進行軟洋菜膠分析，將6孔培養皿之每個孔洞(35 mm)之底部，以2-mL洋菜膠培養基混合物(DMEM，10%(v/v)FCS，0.6%(w/v)洋菜膠)包覆。經底層洋菜膠固化後，將含有2×104
細胞之2-mL上層洋菜膠培養基混合物(DMEM，10%(v/v)FCS，0.3%(w/v)洋菜膠)加入，並於37℃培養4週。將該等培養盤以0.5 mL 0.005%結晶紫進行染色，並以解剖顯微鏡計算細胞聚落數目。
使用帶有聚碳酸酯濾膜8-μm孔徑大小(Corning，英國)之24孔盤Transwell系統進行細胞侵入能力評估。將該膜以MatrigelTM
(BD Pharmingen，NJ，USA)進行包覆。將癌細胞懸浮液，以1×105
細胞密度種於100 μL無血清培養基，種於Transwell上方反應室。下方反應室則以無血清培養基，或帶有10%血清培養基進行沖填。經24小時培養後將培養基移除，並將該濾膜以4%福馬林固定1小時。接著，將濾膜上朝向下方反應室之剩餘細胞以Hoechst 33258(Sigma-Aldrich)進行染色。再以倒立顯微鏡，於100倍倍率下，自5種不同視野觀察及計算移動的癌細胞。
所有動物操作皆按照台北榮民總醫院之國際動物福祉規範進行。將2×105
GBM細胞注入8週齡SCID小鼠(BALB/c株)之腦部紋狀體中。藉由使用顯影發光裝置(LT-9500 Illumatool TLS，裝有激發光源[470 nm]及濾片[515 nm])(Kao et al.,2009)觀察活體內GFP影像。使用卡尺測量腫瘤大小。再以下式計算腫瘤體積：(長度×寬度2
)/2，並以影像Pro-plus軟體進行分析(Kao et al.,2009)。
所有涉及動物之操作皆按照台北榮民總醫院之國際動物福祉規範進行。將8週齡裸鼠(BALB/c株)原位注射不同數量之細胞。接著使用IVIS50動物影像系統(Xenogen Corp.)以進行BLI。由於從標靶部位所發射出來之光子可穿過哺乳動物組織，因此可使用敏感之光化影像系統以從外部偵測該光子並定量。該影像拍攝時間為1分鐘。可使用活體影像軟體(Living Image software)(Xenogen Corp.)以描繪腫瘤位置所呈現之影像，且可以每秒之光子數量定量。使用具有小型正交線圈(mini quadrature coil)(內直徑12cm)之3T MR影像Biospect系統(Bruker)，以每週測量每隻小鼠之腫瘤大小，該小型正交線圈可射頻穿透及接收MR影像訊號(Chiou et al.,2006)。根據下式：(長度×寬度2
)/2，以計算體積，接著以Image-Pro Plus軟體分析。
使用Trizol試劑(Life Technologies，Bethesda，MD，USA)自細胞中萃取所有的RNA，並以Qiagen RNeasy(Qiagen,Valencia，CA，USA)管柱進行純化。以Superscript II RNase H反轉錄酶(Gibco BRL)將所有的RNA進行反轉錄以生成有Cy3-及Cy5-標記(Amersham Biosciences Co.，Piscataway，NJ，USA)之cDNA探針，以分別作為對照組及試驗組樣本。將有標記之探針與含有10,000基因殖株固化cDNA片段之cDNA微陣列進行雜交。分別使用GenePix 4000B陣列掃瞄機(Axon Instruments，Burlingame，Ca，USA)以測量及掃瞄Cy3及Cy5標靶物之螢光強度。使用GenePix Pro 3.0.5.56(Axon Instruments，USA)及GeneSpring GX 7.3.1軟體(Agilent，Palo Alto，CA)進行資料分析。以下詳述如何使用平均連結距離(average-linkage distance)以評估兩組基因表現圖譜間之相似性。對於第三者而言，兩組樣本表現圖譜間的距離差異係藉由比較相對應之平均連結距離(所考量之兩組成員間之所有成對距離(pair-wise distances)(連結)之平均)來評估。對於此種比較中之差異，可藉由結合有關連之平均連結距離之標準差(成對連結之標準差除以連結數之平方根)來估計。使用R程式之標準功能以執行經典多維標度法(multidimensional scaling，MDS)，以提供一視覺印象以使人得知該等不同樣本之間是如何相關連的。
數據皆以平均值±SD方式呈現。視適用情況，使用Student’st
測試或變方分析(analysis of variance，ANOVA)試驗以比較組間之連續變異性。使用卡方檢定(chi-square test)或費雪精確性檢定(Fisher's exact test)以比較類別變項(categorical variable)。使用Kaplan-Meier法以估計存活率，及藉由對數等級檢定(log-rank test)比較。P
<0.05被認為具統計上顯著差異。
多形性神經膠質母細胞瘤(GBM)之預後通常都不佳，即便是經過手術完全切除接著佐以化學放射性治療，仍常於第1年內伴隨著病況快速發展及復發。部分研究指出該等侵略性及復發性GBM可能是由於癌症幹細胞(CSC)(或常稱為腫瘤誘發細胞(TIC))的持續性所造成。近期研究顯示微型核糖核酸(microRNA)可用以調控或活化惡性癌症的CSC特性。然而，微型核糖核酸究竟如何於GMB中調控或產生CSCs，以導致GMB對於傳統療法之抗性及復發，仍不清楚。本發明試著藉由釐清微型核糖核酸於復發性GBM中之角色以探討此一機制。一開始，比較來自4個獨立案例之相同病患之原發性及復發性GBM的miRNA表現圖譜，吾人發現，於原發性及復發性樣本間(總共四種病患組，圖1A)，有22個miRNAs之表現有分歧性(向上或向下表現)。為了進一步評估微陣列數據，收集70個原發性及30個復發性GBM病患樣本(表4)，並藉由定量RT-PCR分析其22個miRNAs表現程度(圖9)。於原發性及復發性GBM間，發現miR142-3p的表現最具顯著不同之差異(圖1B；圖9A)。
表4 GBM病患之臨床特徵
以螢光原位雜交(FISH)偵測出原發性GBM之miR142-3p具高表現量，然而以FISH於復發性GBM腫瘤組織中則無法測得miR142-3p(圖1C)。再者，以北方點墨法分析7組(先前所述之4組加上額外3組)原發性及復發性GBM組織(從7個GBM病患之第一次手術及第二次手術中所切下之腫瘤樣本)之miR142-3p表現。相較於第一次手術取得之樣本(圖1D)，第二次手術所切得之腫瘤樣本之miR142-3p的表現較低，暗示低的miR142-3p表現量與腫瘤復發及發展呈現逆相關性。
經發現miR142-3p之低表現量與GBM復發的相關性後，可推測miR142-3p可能調控GBM細胞之幹細胞性或類CSC性，此可能透過下游與幹細胞機制或標記有關之標的來調控。為了探討miR142-3p可能的下游標的，係使用一軟體篩選策略(Targetscan program,www.targetscan.org)來探究。吾人發現，僅CD133之3’UTR區，(一種穿膜蛋白，已廣為被應用於分離各種癌症類型中之可能的CSC細胞群)高度吻合為miR142-3p之標的。進一步，搜尋NCI60腫瘤資料庫(60種國家癌研所細胞株之基因表現及微型核糖核酸圖譜之資料庫)，且篩選標的之表現量係與miR142-3p呈現負相關性者，結果顯示Sox2(一種轉錄因子，常表現於胚胎幹細胞及多種腫瘤類型之CSCs中)；及ADCY9(AC9)(一種與細胞膜結合的腺苷酸環化酶，於腦部高度表現)；其表現圖譜係與miR142-3p者相反，即：相較於資料庫中其他細胞株，Sox2及AC9於NCI60資料庫之腦部腫瘤細胞株中高度表現，然而miR142-3p表現則為低落的(圖1E)。Sox2、AC9及CD133 mRNA量於70個原發性及30個復發性GBM病患樣本之qRT-PCR分析，顯示miR142-3p在原發性GBM組織中表現較高，而該三種miR142-3p可能之標的的確則會優先表現於復發性GBM組織中(圖1F)。值得注意的是，統計分析該等基因於7組原發性及復發性GBM樣本之mRNA表現，顯示miR142-3p與該三個可能標的間之強烈的負相關性(圖1G)。綜論而言，以上數據指出miR142-3p表現係與Sox2、AC9及CD133之水平及GBM發展或復發呈現逆相關性。
既然miR142-3p與Sox2、AC9、CD133及GBM發展或復發之間為逆相關性，下一個要探討的是，該等分子表現水平與GBM病患之病況之間的關連性。Sox2、AC9、CD133及miR142-3p之表現，分別藉由免疫組織化學染色法及FISH，將來自7個GBM病患以樣本陣列表方式呈現而分析(圖2A，其中一具代表性示例呈現於A圖中)。當與原發性(首次手術)GBM樣本比較時，增加的Sox2、AC9、及CD133之表現量與復發的、幾乎未分化的GBM組織(二次手術)呈現正相關(圖2A及B)。GBM復發性及miR142-3p的表現水平間亦呈現一顯著的負相關性(圖2A及B)，此與先前數據吻合。為了決定Sox2、AC9、CD133與miR142-3p表現水平之預後的重要性，係依照該等基因表現圖譜以Kaplan-Meier存活率分析GBM病患。結果顯示該等病患之AC9或Sox2呈現陽性，但CD133則無呈現陽性時，其存活率下降；然而，相較於具有低度miR142-3p表現之病患，具有高度miR142-3p表現的病患具有較佳的存活率(圖2C；表2)。
再者，相較於具有高度miR142-3p表現及陰性之AC9及Sox2表現的病患，AC9及Sox2呈現陽性且miR142-3p呈現低表現的GBM病患被預測具有較低的存活率(p=0.002，圖2D)。然而，與圖2C結果一致地，CD133對於GBM病患存活率不具指標效果，無論CD133於Sox2-
AC9-
miR142-3p高度表現
及Sox2+
AC9+
miR142-3p低度表現
之病患中是否存在，皆不會改變其存活率(圖2E；p
=0.523：第1組vs第2組；p
=0.314：第3組vs第4組)。於該數據中，CD133之表現係與GBM復發呈現正相關(圖1F及圖2B)，但未與病患存活率呈現正相關(圖2C及圖2E)。綜論之，該等數據指出Sox2+
/AC9+
/miR142-3p低度表現
之指標可做為疾病發展及GBM病患臨床病況之具潛力的預測指標(p
<0.01；圖2D及圖2E)。
miR142-3p及Sox2、AC9、及CD133之間的逆相關性，暗示著可探究Sox2、AC9、及CD133是否可直接被miR142-3p所標靶。構築含有Sox2、AC9、及CD133之野生型(WT)(SEQ ID NOS. 60-62)或其3' UTR區域的依序刪除型(D1-D5)之螢光酵素報信子質體(圖3A至圖3C，右側)。藉由將報信子質體與或不與miR142-3p同時轉染至培養的復發性GBM細胞中以進行螢光酵素報信子試驗，其中該復發性GBM細胞係分離自病患樣本(圖3A至圖3C，左側)。該結果顯示miR142-3p標靶Sox2 3' UTR之3'端附近(圖3A)以及AC9 3' UTR之D1及D2間的區域(圖3B)，即使以Targetscan預測程式(www.targetscan.org)未在Sox2及AC9之3’UTR序列中找到miR142-3p標靶序列。再者，如同Targetscan程式所預測，miR142-3p標靶CD133 3'UTR區域，且D4及D5間之區域對於以miR142-3p為媒介的CD133之表現消除相當重要(p
<0.01；圖3D)。根據此結果，將miR142-3p序列(SEQ ID NO.59)與每個3’UTR之可能區域進行比對，並預測於Sox2、AC9、及CD133中miR142-3p所標靶之區域(圖3F)。接著構築含有野生型(WT)(SEQ ID NOS. 53、55、57)或突變的miR142-3p標靶區域(SEQ ID NOS. 54、56、58)之螢光酵素報信子質體(圖3D，F)。報告試驗顯示miR142-3p抑制野生型標靶位置之螢光酵素活性，但不會抑制突變位置構築之螢光酵素活性(圖3E)，暗示miR142-3p所媒介之Sox2、AC9、及CD133的抑制作用係基於該等被找到之序列。為了確認miR142-3p對於Sox2、AC9、及CD133之抑制作用的相關性，將miR142-3p反義及miR142-3p SPONGE構築體，其係由含有多個、串連之感興趣miRNAs的結合位置之轉錄物所組成的miRNAs之競爭型抑制因子(Ebert et al.,2007)，與野生型或突變型3’UTR報信子共同轉染至復發性GBM細胞中；經以miR142-3p反義及SPONGE構築體處理後，則不再能觀察到抑制作用(圖10)。綜論之，該等數據指出Sox2、AC9、及CD133於復發性GBM細胞中為miR142-3p之直接抑制的標的。
為了分析miR142-3p於調控復發性GBM侵略能力之角色，藉由慢病毒感染來自兩個病患(GBM1及GBM2)的復發性GBM細胞(rGBM)以過量表現miR142-3p。同時亦構築空的載體轉染對照組(rGBM/pLV)。分別以北方墨點法(圖4A下面2個墨點圖)及西方墨點法(圖4A，上面4個墨點圖)分析該等細胞株之異位表現的miR142-3p及內源性Sox2、AC9、及CD133之蛋白量。於該等細胞株之功能性分析顯示，相較於原本的(parental)rGBM及對照組rGBM/pLV細胞，過量表現miR142-3p之rGBM(rGBM/miR142-3p)細胞株具有降低的球體形成能力(圖4B)、較少的軟洋菜膠聚落數目(圖4C)、以及抑制的轉移及侵入能力(圖4D)。吾人進一步於rGBMs中探討miR142-3p調控Sox2、AC9、及CD133之表現與致腫瘤性間的關連性。使用shRNA媒介之基因靜默方式，將衍生自GBM1及GBM2樣本之rGBM細胞中之Sox2、AC9、或CD133專一地剔弱(knocked-down)(圖4E)，並將之進行活體外功能性分析及活體內腫瘤形成試驗。與miR142-3p過量表現結果相似，剔弱研究顯示Sox2或AC9但非CD133之shRNAs(圖4F至G，將shSox2、shAC9、及shCD133與shScr比較；圖12)可明顯抑制rGBM細胞之侵入能力(圖4F)、非貼附性(圖11A至B)、球體形成能力(圖11C至D)、及活體內致腫瘤性(圖4G)。為了判定miR142-3p所調控的Sox2及AC9之向下調控是否確實歸因於該miR142-3p的壓抑效果被抑制，故將Sox2及AC9重新表現於rGBM/miR142-3p細胞中(圖11E)，此導致增強之致腫瘤性(圖11F)。如同預期，該細胞株之Sox2及AC9表現皆已很低(圖11E，中間)，係歸因於miR142-3p的存在，故將Sox2/AC9同時剔弱於此試驗中僅具有很低的影響(圖11F)。由此可推論miR142-3p及Sox2/AC9所媒介之rGBM致腫瘤性為同時發生的事件，且於rGBM中過量表現miR142-3p會部分地，若非全部地，降低腫瘤侵略特性，此係透過抑制Sox2及AC9表現所致。
近期報告指出復發性GBM具有癌症類幹細胞(CSC)特性之潛力且表現出侵略性腫瘤之特徵。基於該等研究，以及根據本發明所發現者，於復發性GBM中miR142-3p表現低而於原發性GBM中表現高，miR142-3p與GBM之癌症幹細胞性間的關係將進一步探討。藉由miRNA SPONGE(Spg)方式，將內源性miR142-3p於原發性GBM細胞(pGBM)中剔弱，該原發性GBM細胞係由如前述之2個相同病患(GBM1及GBM2)所分離而得。於miR142-3p剔弱之原發性GBM(pGBM/Spg)細胞中，其內源性Sox2、AC9、及CD133蛋白表現比原本的pGBM及對照組(pGBM/Scr)細胞較高(圖5A)。接著，藉由球體形成及聚落形成試驗評估pGBM/Spg細胞之自我新生及致腫瘤性能力；該兩種能力於pGBM/Spg中皆比原本的或對照組細胞要高(p
<0.05；圖5B)。吾人亦檢查續代培養物中成功的球體形成，此為評估CSCs持續自我新生能力的一個重要指標，結果顯示從該三個原發性GBM病患樣本(GBM1-3)所建立之所有pGBM/Spg細胞株之自我新生能力，於培養好幾代中皆被保留下來(圖5C)。再者，於pGBM/Spg細胞之轉移/侵入能力亦明顯地較pGBM或pGBM/Scr細胞高(p
<0.05；圖5D)，且於pGBM將miR142-3p剔弱亦可同時發現側群細胞之細胞數目的增加(p
<0.05；圖5E)，此點被認為可能是由於CSCs所造成的。miR142-3p於調控GBM-CSCs之角色可進一步藉由miR142-3p之水平於非CSC-GBM細胞中為高的，但於GBM-CSCs中為低的事實所支持。根據球體形成能力、CD133表面標記表現、以及側群細胞鑑定(圖13)，從7個GBM病患樣本中分離出類CSC之GBM細胞。所有自7個病患所分離之GBM-CSCs展現低度的miR142-3p表現。此外，以生物資訊分析轉錄體標記(transcriptome signature)，指出於pGBM中壓抑miR142-3p，可促進標記朝向間葉幹細胞(MSC)及胚胎幹細胞(ESC；圖5F及圖14)轉變。
接著分析miR142-3P之向下調控是否增加活體內pGBM之腫瘤誘發活性。與不規則的SPONGE轉染對照組比較，於異種移植非SCID小鼠中，吾人發現靜默內源性miR142-3p可增加原發性GBM細胞之腫瘤形成能力(圖5G，GFP訊號代表腫瘤所在位置)。資料顯示於pGBM細胞中SPONGE-miR142-3p增加活體內腫瘤誘發能力10至10,000倍；其中該pGBM細胞係自6個GBM病患所分離(圖5H；圖15)。當將從8號病患所取得之pGBM細胞，以細胞數量為500,000個之多的數目注入NOD-SCID小鼠之紋狀體時，無法形成腫瘤(圖5H，原本的(parental))。然而，以SPONGE將miR142-3p剔弱後，只要用50個之少量細胞數目注入，即可於三個移植的NOD-SCID小鼠中之一個內再生出新的腫瘤；以1000個細胞注入，則三隻小鼠皆會形成新腫瘤(圖5H，miR142-3p SPONGE)。此證據證實於pGBM將miR142-3p向下調控可提供類幹細胞特性以誘發及重新生成新的腫瘤。總而言之，於GBM細胞中維持miR142-3p於一低度的表現量，對於維持CSC特性及活體內腫瘤誘發活性而言十分重要。
相較於原發性GBM細胞，復發性GBM細胞已顯示具有更多之侵略性及間葉特性。Carro等人(2010)證實神經膠質瘤特異性調控網絡與一轉錄的模組有關，其可活化於惡性神經膠質瘤之間葉基因表現。為了探討miR142-3p是否於GBM中主導間葉的轉化，係使用微陣列分析轉錄體圖譜。生物資訊分析結果顯示於rGBM細胞中過量表現miR142-3p，會降低間葉的及誘導神經元的轉錄體，而於pGBM中靜默miR142-3p可誘導間葉的轉錄體(圖6A)。與此資料一致地，多維尺度(MDS)分析顯示將miR142-3p剔弱會將pGBM細胞重新編碼改造朝向間葉標記，然而，將miR142-3p過量表現於rGBM中，將使其從原本朝向間葉改向為原神經元性標記(圖6B)。該數據證實miR142-3p出現與否會決定GBM細胞之幹細胞性標記的特性。
為了探明在miR142-3p依賴性之間葉轉化中Sox2及AC9之參與，將表列之間葉傾向標記(YKL40(亦稱為CHI3L1)、纖維粘連蛋白(fibronectin)、及波形蛋白(vimentin))及膠質纖維酸蛋白(glial fibrillary acidic protein)(GFAP，一種特異性的神經膠細胞系標記)(Carro等人，2010)進行比較。於rGBM細胞中雙重剔弱Sox2/AC-9或過量表現miR142-3p，YKL40、纖維粘連蛋白、及波形蛋白轉錄物皆被減少，而GFAP轉錄物則被增加(圖6C、圖6E)。相反地，於pGBM中將Sox2/AC9過量表現或將miR142-3p靜默，會導致GFAP被壓抑，但提高了間葉傾向標記之表現(圖6D、圖6E)。值得注意的是，當額外同時過量表現Sox2/AC9，rGBM細胞中由miR142-3p所媒介之間葉傾向標記之抑制作用會被增強；反之亦然(圖16A)。使用免疫螢光染色分析衍生自rGBM/miR142-3p移植之免疫功能低下的小鼠之異種移植腫瘤，亦顯示降低的膠原蛋白5a(Col5a，一種GBM之間葉標記)及纖維粘連蛋白的表現水平(圖6F)；以及過量表現Sox2/AC9會恢復Col5a及纖維粘連蛋白之表現(未呈現數據)。miR142-3p-Sox2/AC9調控機制於間葉轉化的角色係藉由傷口癒合移動試驗進一步支持，於該試驗中，miR142-3p所導致rGBM移動力之抑制，會經由同時過量表現Sox2/AC9而恢復，而miR142-3p SPONGE所媒介之增強的pGBM移動力，會藉由將Sox2/AC9雙重剔弱抑制(圖16B-C)。再者，累積存活率分析指出，相較於原本的rGBM移植小鼠，rGBM/miR142-3p移植的免疫功能低下小鼠具有增高的存活率；隨著額外的Sox2/AC9同時過量表現，存活率則會下降(圖6G，上圖)。相反地，由miR142-3p SPONGE媒介的pGBM存活率下降，則可進一步藉由雙重剔弱Sox2/AC9恢復(圖6G，下圖)。總言之，該等結果指出miR142-3p依賴性之GBM細胞間葉轉化可能部分地，如非全部地，透過Sox2及AC9調控。
對於放射性治療具抗性係為在惡性神經膠質瘤中判定癌症幹細胞(CSCs)之主要的臨床要件之一(Bao et al.,2006)。miR142-3p調控GBM之放射線敏感性的方式遂被評估。為了判定放射線敏感性，使用游離輻射(1R)劑量從0到10 Gy以治療復發性GBM，且伴隨著或不伴隨著miR142-3p過量表現。如圖7A顯示，經過IR治療後，沒有以miR142-3p過量表現(rGBM/pLV及原本的rGBM細胞)時，存活的rGBM細胞數目明顯高於該等rGBM/miR142-3p細胞(P<0.01；圖7A)。相反地，相較於原本的(parental)及載體對照組(圖7B)，以SPONGE miR142-3p處理之原發性GBM細胞具有較高的存活率，以及亦具有較高程度的抗放射線性。為了進一步評估miR142-3p於含有rGBM腫瘤之免疫功能低下小鼠之放射線敏感性效果，將rGBM/miR142-3p及rGBM/pLV注射入小鼠中，並以4 Gy IR治療，並以GFP陽性區域分析腫瘤大小(圖7C)。結果顯示，相較於rGBM/pLV腫瘤，經放射性治療後，miR142-3p減低腫瘤大小。含有rGBM/pLV腫瘤或rGBM/miR142-3p腫瘤之小鼠係以或不以IR治療，且監控腫瘤生長6週(圖7D)。經過移植6週後(rGBM/pLV)，rGBM細胞形成一大型的腦部腫瘤；而經IR治療對於腫瘤生長僅具有有限的抑制效果(rGBM/pLV/IR)。相反地，過量表現miR142-3p可大幅度地抑制腫瘤生長(rGBM/miR142-3p)；且伴隨著額外的IR治療，腫瘤大小幾乎被維持在難以偵測到的程度(rGBM/miR142-3p/IR)。此結果不僅確認了miR142-3p對於腫瘤生長之抑制作用的角色，亦呈現miR142-3p及IR療法的協同作用。再者，為了探討Sox2及AC9於miR142-3p所媒介的放射線敏感性之參與性，將小鼠移植pGBM/Scr、pGBM/Spg、或pGBM/Spg+shSox2/shAC9細胞，並以IR或不以IR治療，再以MRI影像監控(圖7E-F)。結果顯示，相較於對照組pGBM/Scr細胞之放射線敏感性，即使伴隨IR治療，將內源性miR142-3p(pGBM/Spg)靜默明顯地促進腫瘤生長(圖7F)。當額外將Sox2及AC9雙重剔弱(pGBM/Spg/shSox2+shAC9)後，pGBM/Spg之放射線敏感性增加了。再者，雙重剔弱Sox2及AC9對放射線敏感性之恢復功效，亦可於活體內pGBM腫瘤誘發能力試驗中被觀察到(圖7G)。miR142-3p-SPONGE在pGBM細胞所媒介之提高的腫瘤誘發能力，會被雙重剔弱Sox2/AC9所抑制，且此抑制作用經IR治療後更加明顯。綜論之，該等資料指出miR142-3p使得GBM細胞對於IR敏感化，且Sox2及AC9皆參與此一作用。
以往，藉由腺病毒載體或注射以鎖核酸(LNA)或磷酸硫代二酯修飾的骨架所構築之miRNAs進行miRNA之基因遞送，在腫瘤發展上展現一可靠的抑制作用(Patrick et al.,2010;Zhang et al.,2010)。經LNA修飾的miR-142-3p寡核苷酸於抑制復發性GBM腫瘤之潛在療效遂被分析(圖8A)。將LNA修飾的突變的或野生型miR-142-3p(分別為Mut LNA-miR142-3p及LNA-miR142-3p)注射至腫瘤所在位置5天前，先將rGBM-GFP細胞注入小鼠之腦部的紋狀體中。相較於以Mut LNA-miR142-3p注射之rGBM-GFP腫瘤，根據GFP影像，LNA-miR142-3p減少了腫瘤體積(圖8A)；且LNA-miR142-3p對於腫瘤大小之抑制效果亦可於衍生自GBM1及GBM2之rGBM腫瘤上被觀察到(圖8B)。為了分析腫瘤縮小是否歸因於腫瘤轉變成其他神經膠質瘤細胞類型所致，將以Mut LNA-miR142-3p或LNA-miR142-3p處理的小鼠之腦部腫瘤樣本切片進行染色，結果顯示於LNA-miR142-3p處理的腫瘤中，波形蛋白、及纖維粘連蛋白之表現降低(圖8C)。此病理分析暗示經LNA-miR142-3p注射會降低rGBM腫瘤之間葉特性。再者，間葉標記(YKL40、神經巢蛋白、纖維粘連蛋白(FN)、波形蛋白(VM))及幹細胞性基因(Oct4、Sox2、Nanog、Klf4)的表現水平明顯地於以LNA-miR142-3p處理之rGBM腫瘤中被抑制了(圖8D)。活體內之LNA-miR142-3p的放射線敏感性功效接著被分析；相較於Mut LNA-miR142-3p加上IR治療的腫瘤，經LNA-miR142-3p及IR治療後，可觀察到rGBM腫瘤體積明顯降低(圖8E)。值得注意的是，雖然單獨以LNA-miR142-3p處理時似乎可做為一種腫瘤抑制劑(*p<0.05；圖8E，右圖中的白線)，但LNA-miR142-3p及IR之協同作用將可更進一步抑制腫瘤體積(#p<0.05；圖8E右圖)。綜論之，經微脂體包埋的LNA-修飾的miR142-3p寡核苷酸注射，可使顱內rGBM細胞轉變為較不具惡性的神經膠質瘤細胞，大幅度地降低間葉特性及明顯的改善放射線敏感性。
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為了闡述本發明之目的，將具體實施例以較佳方式呈現於圖式中。然而，吾人應理解的是，本發明並非侷限於所示之較佳具體實施例。
於圖式中：
圖1顯示miR142-3p被做為復發性GBM指標之一反向的預測因子。
圖2顯示低量miR142-3p以及高量Sox2及AC9表現水平之情況，其為復發性GBM及低總存活率之預測指標。
圖3顯示miR-142-3p直接標靶GBM細胞中之Sox2、ADCY9、及CD133 3' UTRs區域。
圖4顯示miR142-3p弱化GBM細胞之自我新生能力及致腫瘤性。
圖5顯示miR142-3p SPONGE恢復原發性GBM細胞之幹細胞性及腫瘤誘發能力。
圖6顯示miR142-3p及Sox2/AC9表現可調控GBM之間葉轉化以及原神經(proneuronal)轉變。
圖7顯示miR142-3p、Sox2及AC9調控GBM細胞之腫瘤誘發能力及放射線敏感性。
圖8顯示miR142-3p調控復發性GBM細胞之致腫瘤性及放射線之敏感性。
圖9顯示於原發性(N=70)及復發性(N=30)GBM病患之22個miRNAs表現量的定量RT-PCR分析，包括於復發性GBM中miR223、miR451、miR181a、miR9、miR142-3p、miR195、miR145、miR181c、miR132、及miR497之下降的表現量，其中以miR142-3p之p
值(p=0.0016)，於原發性(第一次發生)及復發性(第二次發生)GBM之間，最為顯著(此處數據顯示三次獨立試驗之平均值±SD的結果)。
圖10顯示將Sox2(A)、AC9(B)、及CD133(C)之野生型(wild-type，WT)或突變的(mutated，mut)3’UTR報信子，與(或不與)對miR142-3p反義的miRNA及SPONGE一起共同轉染，其中測量每一個報信子質體之螢光酵素(luciferase)活性，其顯示無論是突變的3’UTR、反義的miR142-3p、或SPONGE-miR142-3p皆可消除miR142-3p對於Sox2、AC9、及CD133 3’UTR之抑制作用(*p<0.05)。
圖11顯示Sox2及AC9在GBM細胞之致腫瘤性的探討。(A、B、C、D)建立pGBM/pLV、pGMB/Sox2、pGBM/AC9、rGBM/shScr、rGBM/shSox2、及rGBM/shAC9細胞株，並將之進行軟洋菜膠細胞聚落形成試驗(A、B)及聚球形成試驗(C、D)；(E)過量表現的Sox2或AC9可增加pGBM之非貼附性及自我新生的能力，而Sox2或AC9之剔弱則會減少該rGBM的兩種能力；該rGBM/miR142-3p細胞進一步被用以生成rGBM/miR142-3p/shSox2+shAC9及rGBM/miR142-3p/Sox2+AC9細胞株；並藉由西方墨點法分析Sox2及AC9之蛋白量；(F)測量異種移植之腫瘤大小，其中該異種移植腫瘤係源自植入所指細胞之老鼠。Sox2/AC9共同過量表現會增加rGBM/miR142-3p腫瘤的大小(*p<0.05)。
圖12顯示CD133於GBM細胞中之致腫瘤性的研究：(A)pGBM/CD133細胞株係自病患GBM1及GBM2之細胞衍生而得；並藉由西方墨點法評估於每個細胞株之CD133蛋白表現量；(B、C、D)將pGBM/CD133、rGBM/shCD133及其對照組細胞進行軟洋菜膠聚落形成試驗(B)、聚球試驗(C)及細胞侵襲/轉移試驗(TransWell invation/migration)(D)；CD133之存在與否對於兩種pGBM細胞株之非貼附性、自我新生以及侵襲/轉移能力僅有輕微的影響；(E)評估經異種移植rGBM/shScr及rGBM/shCD133之NOD-SCID小鼠之腦紋狀體中腫瘤生長及體積。經觀察無顯著差異；以及(F)經植入所指細胞的NOD-SCID小鼠的腫瘤體積，於移植後28天測量之；其中於rGBM/shScr及rGBM/shCD133腫瘤之間，以及於pGBM/pLV及pGBM/CD133腫瘤之間，皆無顯著差異。
圖13顯示自7個GBM病患分離之癌類幹細胞的miR142-3p表現情形：(A)左圖：兩個個別的GBM細胞株(GBM1及GBM2)的球形體之顯微鏡影像，其中於右圖：顯示於7個病患之球形體形成GBMs中之miR142-3p表現量為降低的；(B)於左圖：miR142-3p之內源性表現，係藉由FISH(螢光原位雜交(fluorescent in situ hybridization)；綠色：代表miR142-3p有表現；GBM1)測量。右圖：於7個病患組中，相較於CD133-
GBM細胞，結果顯示於CD133陽性(CD133+
)GBM細胞之miR142-3p表現為向下調控的；(C)左圖：將GBM細胞之側群細胞(Side populations)分離，並藉由細胞分類器(GBM1)分離。右圖：7個病患中側群細胞(SP)為陽性的GBM細胞之降低的miR-142-3p表現。*p<0.05。
圖14顯示於胚胎幹細胞(embryonic stem cell，ESCs)及GBMs中轉錄體圖譜之生物資訊分析結果：多維度分析說明了於ESCs、rGBM、具有miR142-3p之rGBM、pGBM、及具有miR142-3p SPONGE之pGBM之間的平均譜系轉錄體距離(average lineage transcriptome distance)。
圖15顯示分離自5個病患之pGBM細胞經具有SPONGE之miR142-3p剔弱方法處理後的腫瘤誘發能力之評估；以及分別將50、100、1000、及10000之細胞數量同位(orthotopically)注射入所指的SCID小鼠的腦紋狀體中；miR142-3p SPONGE之腫瘤誘發能力，於pGBM細胞中藉由Sox2/AC9雙重基因靜默被進一步弱化。
圖16顯示Sox2及AC9涉及miR142-3p所調控的間葉轉化：(A)將pGBM/Scr、pGBM/Spg、pGBM/Spg/shSox2+shAC9、rGBM/pLV、rGBM/miR142-3p、及rGBM/miR142-3p/Sox2+AC9細胞進行qRT-PCR，分析間葉傾向之標記(YKL40、波形蛋白(vementin)、纖維粘連蛋白(fibronectin))的表現情形；(B)傷口癒合移動試驗係以所指細胞進行；(C)計算不同組的移動活性，且以相對值呈現於表中；*: p<0.05，pLVv.s
. pLV-miR142/Vector；#: p<0.05，pLV-miR142/Vectorv.s
. pLV-miR142/Sox2+AC9。
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Claims (10)
- 一種將miR142-3p用於製造治療多形性神經膠質母細胞瘤之醫藥組合物的用途。
- 如申請專利範圍第2項之用途，其中該醫藥組合物係藉由立體定位裝置投與至腫瘤所在位置。
- 如申請專利範圍第1項之用途，其中該多形性神經膠質母細胞瘤為復發性多形性神經膠質母細胞瘤。
- 如申請專利範圍第1項之用途，其中該miR142-3p係由核酸所編碼。
- 如申請專利範圍第4項之用途，其中該核酸係位於選自由質體、黏質體、噬菌質體、或病毒所組成之群組的載體上。
- 如申請專利範圍第1項之用途，其中該miR142-3p為具有經LNA-及硫代磷酸酯-修飾骨架之修飾的微型核糖核酸(microRNA)。
- 如申請專利範圍第6項之用途，其中該修飾的微型核糖核酸被包埋於微脂體中。
- 如申請專利範圍第1項之用途，其中該miR142-3p於多形性神經膠質母細胞瘤內抑制目標基因包含CD133、Sox2、及AC9之表現。
- 如申請專利範圍第8項之用途，其中該miR142-3p係直接與目標基因之mRNA的3’端非轉譯區(3’UTR)連結。
- 如申請專利範圍第1項之用途，其增加多形性神經膠質母細胞瘤細胞對於放射性療法敏感性。
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| WO2014111561A1 (en) * | 2013-01-21 | 2014-07-24 | Deutsches Krebsforschungszentrum | Serum mirna-142-3p as prognostic cancer marker |
| JP2017522555A (ja) * | 2014-06-26 | 2017-08-10 | インスティテュート フォー システムズ バイオロジーＩｎｓｔｉｔｕｔｅ Ｆｏｒ Ｓｙｓｔｅｍｓ Ｂｉｏｌｏｇｙ | 多形膠芽腫（ｇｂｍ）のためのマーカー及び治療指標 |
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| CN108893534A (zh) * | 2018-06-26 | 2018-11-27 | 华中科技大学鄂州工业技术研究院 | 泛癌症诊断标记物 |
| CN110452982A (zh) * | 2019-05-08 | 2019-11-15 | 中山大学孙逸仙纪念医院 | 乳腺癌循环肿瘤细胞miRNA和EMT标志物联合检测试剂盒及其应用 |
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| CN102813940A (zh) | 2012-12-12 |
| EP2505645B1 (en) | 2016-08-17 |
| TW201250005A (en) | 2012-12-16 |
| US20130108691A1 (en) | 2013-05-02 |
| US9107934B2 (en) | 2015-08-18 |
| CN102813940B (zh) | 2016-03-02 |
| US8404437B2 (en) | 2013-03-26 |
| EP2505645A1 (en) | 2012-10-03 |
| US20120255043A1 (en) | 2012-10-04 |
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