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      MicroRNA-21調(diào)控乳腺癌對吉西他濱耐藥的機(jī)制

      2015-01-04 11:21:51
      中國癌癥雜志 2015年5期
      關(guān)鍵詞:親代吉西他濱吉西

      復(fù)旦大學(xué)附屬腫瘤醫(yī)院腫瘤內(nèi)科,復(fù)旦大學(xué)上海醫(yī)學(xué)院腫瘤學(xué)系,上海 200032

      MicroRNA-21調(diào)控乳腺癌對吉西他濱耐藥的機(jī)制

      吳振華,陶中華,張劍,解婕,胡夕春

      復(fù)旦大學(xué)附屬腫瘤醫(yī)院腫瘤內(nèi)科,復(fù)旦大學(xué)上海醫(yī)學(xué)院腫瘤學(xué)系,上海 200032

      背景與目的:以吉西他濱為基礎(chǔ)的聯(lián)合用藥在轉(zhuǎn)移性乳腺癌中展示出很好的臨床療效和安全性,但耐藥的出現(xiàn)導(dǎo)致治療失敗。MicroRNA是一類非編碼小分子RNA,起到類似癌基因或抑癌基因的作用。雖然腫瘤中關(guān)于化療藥物耐藥的機(jī)制報道很多,但microRNA異常表達(dá)與耐藥之間的關(guān)系及機(jī)制還不十分清楚。本研究旨在探討microRNA-21在乳腺癌吉西他濱耐藥中的作用及其可能機(jī)制。方法:采用低濃度持續(xù)誘導(dǎo)MDA-MB-231細(xì)胞的方式構(gòu)建人乳腺癌耐吉西他濱細(xì)胞株,藥物敏感性差異達(dá)10倍以上,繼而通過實(shí)時熒光定量PCR(real-time PCR,RT-PCR)、CCK-8、蛋白[質(zhì)]印跡法(Western blot)、轉(zhuǎn)染、劃痕和Transwell等實(shí)驗(yàn)分別檢測microRNA-21對藥物的敏感性及上皮-間充質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)標(biāo)志物的影響。結(jié)果:在乳腺癌吉西他濱耐藥細(xì)胞株中存在EMT現(xiàn)象,相比親代細(xì)胞,microRNA-21呈高表達(dá),并與吉西他濱敏感性呈負(fù)相關(guān)。轉(zhuǎn)染microRNA-21的抑制劑和mimic可以分別下調(diào)和上調(diào)microRNA-21表達(dá),同時EMT現(xiàn)象和藥物敏感性也發(fā)生了相應(yīng)的變化。結(jié)論:MicroRNA-21可能通過誘導(dǎo)腫瘤細(xì)胞的EMT發(fā)生而介導(dǎo)乳腺癌對吉西他濱的耐藥。

      MicroRNA-21;乳腺癌;吉西他濱耐藥;上皮-間充質(zhì)轉(zhuǎn)化

      吉西他濱是一種核苷類似物,對乳腺癌等實(shí)體腫瘤具有廣譜的抗腫瘤活性。對早期治療失敗的局部復(fù)發(fā)或遠(yuǎn)處轉(zhuǎn)移的晚期乳腺癌患者,吉西他濱常與其他化療藥物相聯(lián)用,如與紫杉醇(GT方案)或順鉑(GP方案)聯(lián)用作為晚期一線用藥并顯示出很好的臨床療效[1]。但經(jīng)治療后仍有一大部分患者出現(xiàn)耐藥,導(dǎo)致治療失敗。在乳腺癌中吉西他濱耐藥的機(jī)制報道尚不多見,具體機(jī)制有待進(jìn)一步闡明。

      上皮-間充質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)是指從具有極性的上皮細(xì)胞轉(zhuǎn)換成具有活動能力的一個過程,細(xì)胞形態(tài)發(fā)生改變并且變得更容易游走。其主要特征為細(xì)胞上皮標(biāo)志物E-cadherin表達(dá)減少以及間充質(zhì)標(biāo)志物如Vimentin、N-cadherin表達(dá)增加[2]。最近越來越多的研究表明,EMT不僅在胚胎發(fā)育中發(fā)揮著重要作用,而且也與腫瘤的轉(zhuǎn)移和耐藥有關(guān)[3-4],并且受到microRNA的調(diào)控[5-6]。

      MicroRNA是長度在18~25個核苷酸的內(nèi)源性非編碼小分子RNA,具有轉(zhuǎn)錄后基因調(diào)控功能[7]。近年來研究表明,腫瘤遷移、侵襲、黏附和EMT等多個腫瘤轉(zhuǎn)移關(guān)鍵步驟中均有microRNA參與調(diào)控,起到類似癌基因或抑癌基因的作用。MicroRNA-21作為第一批在人類基因組中發(fā)現(xiàn)的microRNA,現(xiàn)已被證實(shí)在許多腫瘤(如胃癌、腸癌和乳腺癌等)中常過度激活[8],起到類似癌基因的作用,并與腫瘤的侵襲和轉(zhuǎn)移有關(guān)。近年來許多研究顯示,microRNA-21的表達(dá)與藥物的敏感性相關(guān)[9],但在乳腺癌中尚未有microRNA-21與吉西他濱敏感性的報道。本研究發(fā)現(xiàn),在乳腺癌中microRNA-21可能通過誘導(dǎo)腫瘤細(xì)胞的EMT發(fā)生而介導(dǎo)吉西他濱的耐藥。

      1 材料和方法

      1.1 材料

      人乳腺癌MDA-MB-231細(xì)胞株購自美國典型培養(yǎng)物保藏中心(American Type Culture Collection,ATCC)。胎牛血清FBS、L-15培養(yǎng)基、磷酸鹽緩沖液(phosphate buffer solution,PBS)和0.25%胰蛋白酶購自美國Gibco公司;二甲亞砜(DMSO)、四甲基乙二胺(TEMED)和蛋白酶抑制劑購自美國Sigma公司,磷酸酶抑制劑購自羅氏公司,結(jié)晶紫染色液、RIPA 細(xì)胞裂解液和BCA 蛋白濃度測定試劑盒購自上海碧云天生物技術(shù)有限公司;細(xì)胞計(jì)數(shù)試劑盒(cell counting kit-8,CCK-8)試劑盒購自美國東仁化學(xué)科技(上海)有限公司;Transwell小室和Matrigel膠購自于美國BD公司;吉西他濱(健擇,GEMZAR)粉末購自法國禮來公司(200 mg,貨號:C096711A),PBS稀釋配成不同濃度后于-20 ℃保存;microRNA-21 qRT-PCR正反向及逆轉(zhuǎn)錄引物、microRNA-21 mimic和抑制劑(inhibitor)均購自廣州銳博生物科技有限公司。LipofectamineTM2000購自美國Invitrogen公司。一抗:E-cadherin(兔源單抗,貨號:3195P)、Vimentin(兔源單抗,貨號:5741P)和β-actin(鼠源單抗,貨號:12262S)抗體購自美國Cell Signaling公司,Twist(兔源單抗,貨號:ab152775)抗體購自英國Abcam公司;二抗:HRP-羊抗小鼠IgG和HRP-羊抗兔IgG購自上海威奧生物科技有限公司。

      1.2 方法

      1.2.1 細(xì)胞培養(yǎng)

      將人乳腺癌MDA-MB-231及其耐藥細(xì)胞株置于含有10%胎牛血清的L-15培養(yǎng)基中,在37 ℃、CO2體積分?jǐn)?shù)為5%的條件下培養(yǎng),2~3 d傳代1次。

      1.2.2 細(xì)胞轉(zhuǎn)染

      實(shí)驗(yàn)所涉及的細(xì)胞以適當(dāng)密度傳代鋪板,待融合至50%~70%時按照LipofectamineTM2000說明書轉(zhuǎn)染microRNA-21 mimic及抑制劑,轉(zhuǎn)染24~72 h后完成相關(guān)的Loss and Gain功能實(shí)驗(yàn)。

      1.2.3 實(shí)時熒光定量PCR(real-time PCR,RTPCR)

      利用TRIzol(Invitrogen)提取細(xì)胞中的總RNA,隨后用PrimeScript RT Reagent試劑盒[購自寶生物工程(大連)有限公司]將RNA反轉(zhuǎn)錄成cDNA。按照產(chǎn)品說明書再用SYBR? Premix Ex Taq? Ⅱ?qū)DNA模板進(jìn)行RT-PCR。

      1.2.4 細(xì)胞形態(tài)學(xué)觀察

      選取細(xì)胞貼壁生長48 h時為記錄時刻,在倒置顯微鏡下對細(xì)胞的形態(tài)進(jìn)行拍照觀察。

      1.2.5 CCK-8法檢測藥物敏感性

      在96孔板中每孔接種100 μL細(xì)胞懸液,細(xì)胞總數(shù)為5×103個。在37 ℃、CO2體積分?jǐn)?shù)為5%的細(xì)胞培養(yǎng)箱中溫育24 h,待細(xì)胞貼壁后加入10 μL不同濃度的吉西他濱,每一劑量組設(shè)5個平行孔。培養(yǎng)48 h后,吸凈上清液后每孔加入110 μL用細(xì)胞培養(yǎng)基按10∶1 比例稀釋的CCK-8溶液,37 ℃避光溫育1~4 h后自動酶標(biāo)儀測量562 nm波長下每孔的吸光度(D)值。上述實(shí)驗(yàn)重復(fù)3次,計(jì)算各組細(xì)胞在不同藥物濃度下的細(xì)胞活力。

      1.2.6 細(xì)胞劃痕實(shí)驗(yàn)

      細(xì)胞消化后以適當(dāng)?shù)拿芏冉臃N到6孔板中,待密度融合至90%時,用白色槍頭劃“井”字線,分別在0和24 h時的倒置顯微鏡下拍照。

      1.2.7 細(xì)胞侵襲實(shí)驗(yàn)

      用50 mg/L Matrigel 1∶6稀釋液包被Transwell小室基底膜上室面,每孔50 μL。置于37 ℃培養(yǎng)箱中4 h使膠聚合成凝備用。將MDA-MB-231及其耐藥細(xì)胞胰酶消化后,107×g離心5 min后棄上清液,加入無血清細(xì)胞培養(yǎng)基進(jìn)行重懸和細(xì)胞計(jì)數(shù)。兩組細(xì)胞分別取2.5×104個/200 μL單細(xì)胞懸液加入Transwell上室,另在Transwell下室中加入600 μL含10% FBS的細(xì)胞培養(yǎng)基。在37 ℃細(xì)胞培養(yǎng)箱中培養(yǎng)24 h。去除小室中的培養(yǎng)基,用棉簽輕輕擦去上層的細(xì)胞,置入預(yù)冷的4%多聚甲醛固定20 min。固定后用結(jié)晶紫進(jìn)行染色25~30 min后,用PBS輕輕洗兩遍,倒置晾干。在倒置顯微鏡下進(jìn)行拍照計(jì)數(shù),選取5(中心+4個象限)視野進(jìn)行拍照計(jì)數(shù),重復(fù)3遍,統(tǒng)計(jì)分析。

      1.2.8 蛋白[質(zhì)]印跡法(Western blot)檢測

      選取對數(shù)生長期的細(xì)胞,PBS洗滌2次,按細(xì)胞蛋白裂解液說明書操作提取細(xì)胞蛋白。BCA法測定各組細(xì)胞蛋白濃度,按每泳道20 μg蛋白上樣,5%濃縮膠60 V,10%分離膠120 V,電泳分離約150 min。經(jīng)100 V 120 min濕轉(zhuǎn)至PVDF膜上。10%脫脂奶粉室溫封閉90 min,分別加入一抗E-cadherin(1∶1 000)、Vimentin(1∶1 000)、Twist(1∶1 000)和內(nèi)參β-actin(1∶4 000),分別于4 ℃溫育過夜。TBST洗膜3次,每次5 min,HRP偶聯(lián)的二抗(1∶2 000)室溫溫育2 h,TBST洗膜3次,每次5 min。用ECL發(fā)光液曝光顯影。

      1.3 統(tǒng)計(jì)學(xué)處理

      采用Graph Pad Prism 5.0軟件進(jìn)行統(tǒng)計(jì)分析,雙邊t檢驗(yàn)方法檢驗(yàn)組間差異。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

      2 結(jié) 果

      2.1 人乳腺癌MDA-MB-231耐吉西他濱細(xì)胞株的建立與鑒定

      對人乳腺癌MDA-MB-231細(xì)胞株進(jìn)行低濃度的吉西他濱持續(xù)誘導(dǎo),具體誘導(dǎo)程序?yàn)榧魉麨I連續(xù)處理1周后,正常培養(yǎng)液處理1周,吉西他濱起始濃度為12 nmol/L,依次遞增,最后穩(wěn)定在60 μmol/L,整個建立的過程超過1年[10]。采用CCK-8法測定親代細(xì)胞MDAMB-231和耐藥細(xì)胞株對吉西他濱的敏感性,耐藥細(xì)胞株對吉西他濱的敏感性明顯低于親代細(xì)胞(圖1)。

      圖1 CCK-8檢測MDA-MB-231親代和耐藥細(xì)胞對吉西他濱的敏感性Fig. 1 CCK-8 assay was used to detect chemosensitivity in parent and gemcitabine resistant MDA-MB-231 cell lines

      2.2 人乳腺癌MDA-MB-231耐吉西他濱細(xì)胞株中存在EMT現(xiàn)象

      在建立MDA-MB-231吉西他濱耐藥細(xì)胞株的過程中,發(fā)現(xiàn)細(xì)胞的形態(tài)由親代的短棒狀變?yōu)槟退幍拈L梭狀,細(xì)胞的表型發(fā)生了改變。本研究進(jìn)一步從細(xì)胞的遷移、侵襲能力以及EMT的指標(biāo)來探討耐藥細(xì)胞中是否發(fā)生了EMT。劃痕及Transwell實(shí)驗(yàn)結(jié)果表明,耐藥細(xì)胞與親代細(xì)胞相比,遷移和侵襲能力增強(qiáng)。同時Western blot檢測結(jié)果揭示,在耐藥細(xì)胞中,間充質(zhì)表型標(biāo)志物波形蛋白Vimentin和轉(zhuǎn)錄因子Twist過度表達(dá),而上皮標(biāo)志物E-cadherin表達(dá)降低。這些均表明耐藥細(xì)胞株中發(fā)生了EMT(圖2)。

      2.3 MicroRNA-21與吉西他濱的敏感性呈負(fù)相關(guān)

      通過RT-PCR檢測發(fā)現(xiàn)microRNA-21在耐藥細(xì)胞中過度表達(dá),約為親代的2.7倍(圖3A)。Loss and Gain功能實(shí)驗(yàn)發(fā)現(xiàn),在親代細(xì)胞中通過LipofectamineTM2000瞬時轉(zhuǎn)染microRNA-21 mimic,使microRNA-21過度表達(dá),發(fā)現(xiàn)對吉西他濱的敏感性下降(圖3B、C)。而在耐藥細(xì)胞株中轉(zhuǎn)染microRNA-21抑制劑后,發(fā)現(xiàn)對吉西他濱的敏感性增強(qiáng)(圖3D、E),表明microRNA在乳腺癌中與吉西他濱的敏感性呈負(fù)相關(guān)。

      2.4 MicroRNA-21調(diào)控乳腺癌吉西他濱耐藥中EMT現(xiàn)象

      通過Loss and Gain功能實(shí)驗(yàn),使親代細(xì)胞中microRNA-21過表達(dá)而在耐藥細(xì)胞中低表達(dá)。Western blot檢測EMT相關(guān)指標(biāo)的變化,結(jié)果顯示,在親代細(xì)胞中轉(zhuǎn)染microRNA-21 mimic后,E-cadherin表達(dá)降低而波形蛋白Vimentin和轉(zhuǎn)錄因子Twist表達(dá)增加,誘導(dǎo)EMT發(fā)生。在耐藥細(xì)胞中使microRNA-21表達(dá)下調(diào)后,E-cadherin表達(dá)上調(diào)、Vimentin下調(diào)、Twist表達(dá)下調(diào),發(fā)生間充質(zhì)-上皮轉(zhuǎn)化(mesenchymal-epithelial transition,MET)現(xiàn)象(圖4)。由此可以推斷microRNA-21調(diào)控乳腺癌中吉西他濱耐藥相關(guān)的EMT。

      圖2 耐藥細(xì)胞中發(fā)生EMT現(xiàn)象Fig. 2 EMT was involved in gemcitabine resistant breast cancer cells

      3 討 論

      乳腺癌已成為當(dāng)今嚴(yán)重威脅女性身心健康的惡性腫瘤,在我國乳腺癌的發(fā)病率呈逐年遞增的趨勢。研究發(fā)現(xiàn)約90%的早期乳腺癌患者最終發(fā)展成晚期乳腺癌并發(fā)生多藥耐藥[3]。我們臨床試驗(yàn)研究已證實(shí)吉西他濱與順鉑聯(lián)用作為轉(zhuǎn)移性三陰性乳腺癌晚期一線用藥顯示出很好的臨床療效和安全性[1]。但經(jīng)治療后仍有一大部分患者出現(xiàn)復(fù)發(fā)轉(zhuǎn)移,導(dǎo)致治療失敗。因此,探討乳腺癌中吉西他濱耐藥的機(jī)制以及如何逆轉(zhuǎn)耐藥具有重要的理論意義和臨床應(yīng)用價值。

      吉西他濱是一種核苷類似物,屬于抗代謝類腫瘤藥物,其本身并不具有抗腫瘤作用,而是通過代謝產(chǎn)物而產(chǎn)生細(xì)胞毒性作用。其轉(zhuǎn)運(yùn)途徑及代謝過程中酶活性的異常均會引起其耐藥[11]。目前已報道與吉西他濱耐藥相關(guān)的機(jī)制主要有:核轉(zhuǎn)運(yùn)相關(guān)蛋白hENT1表達(dá)減少[12],細(xì)胞膜上多重耐藥相關(guān)蛋白5(MRP5,ABCC5)表達(dá)增加[13-14],作用靶點(diǎn)RR酶活性增強(qiáng)[15],脫氧胞苷激酶(dCK)活性降低[16],PI3K-Akt促生成信號通路的過度激活及凋亡通路NF-κB的激活[14]。此外,腫瘤干細(xì)胞和EMT現(xiàn)象也參與吉西他濱耐藥的過程。而在乳腺癌中,吉西他濱的耐藥機(jī)制報道尚不多見,僅報道與RRM1[17]和NF-κB[18]信號通路的激活有關(guān),其具體機(jī)制有待進(jìn)一步闡明。

      圖3 MicroRNA-21表達(dá)與吉西他濱敏感性呈負(fù)相關(guān)Fig. 3 MicroRNA-21 was inversely correlated with gemcitabine sensitivity

      圖4 MicroRNA-21調(diào)控EMT標(biāo)記物的表達(dá)Fig. 4 MicroRNA-21 mediated the expression of EMT associated markers

      本研究通過低濃度持續(xù)誘導(dǎo)的方式構(gòu)建了人乳腺癌MDA-MB-231耐吉西他濱體外模型,來進(jìn)一步探討乳腺癌中吉西他濱耐藥的可能機(jī)制。在建立耐藥細(xì)胞系過程中,發(fā)現(xiàn)細(xì)胞的表型發(fā)生了改變,由短棒狀變?yōu)殚L條不規(guī)則狀,同時轉(zhuǎn)移和侵襲能力增強(qiáng)。Western blot檢測結(jié)果也顯示,耐藥細(xì)胞中上皮標(biāo)志物E-cadherin表達(dá)明顯降低,間質(zhì)表型標(biāo)志物Vimentin表達(dá)增加,轉(zhuǎn)錄因子Twist被激活,證實(shí)在耐藥細(xì)胞中發(fā)生了EMT現(xiàn)象。最近越來越多的研究表明,EMT與藥物的耐藥及腫瘤轉(zhuǎn)移有關(guān),如Xia等[19]發(fā)現(xiàn)在肝癌細(xì)胞中EMT與索拉菲尼耐藥有關(guān),Kitamura等[20]發(fā)現(xiàn)在肺癌中EMT與吉非替尼耐藥有關(guān)。因此,我們推測在乳腺癌吉西他濱耐藥細(xì)胞中EMT現(xiàn)象與吉西他濱的耐藥有關(guān)。

      MicroRNA-21是非編碼小分子RNA,在許多腫瘤中常過度激活,起到癌基因的作用。有研究表明,在乳腺癌中microRNA-21可以作為區(qū)別浸潤性癌和非浸潤性癌的分子標(biāo)志[21],但是尚無與吉西他濱敏感性的報道。在本實(shí)驗(yàn)中發(fā)現(xiàn),在耐藥細(xì)胞中microRNA-21高表達(dá),同時Loss and Gain功能實(shí)驗(yàn)證實(shí),microRNA-21表達(dá)與吉西他濱的敏感性呈負(fù)相關(guān)。最近許多文獻(xiàn)報道m(xù)icroRNA調(diào)控著EMT的表達(dá),如microRNA-21 6/217調(diào)控在肝癌中的EMT現(xiàn)象[19],由此我們假設(shè),microRNA-21可能調(diào)控著乳腺癌吉西他濱耐藥中的EMT。當(dāng)在親代細(xì)胞中轉(zhuǎn)染microRNA-21 mimic后,E-Cadherin表達(dá)下降,Vimentin和Twist表達(dá)上調(diào),發(fā)生EMT。而在耐藥細(xì)胞中使microRNA-21表達(dá)下調(diào)之后,E-Cadherin表達(dá)上調(diào),Vimentin和Twist表達(dá)下降,發(fā)生MET。由此可見,microRNA-21調(diào)節(jié)著耐藥細(xì)胞中的EMT現(xiàn)象。

      綜上所述,EMT可能是乳腺癌中吉西他濱耐藥的機(jī)制之一,而microRNA-21可能通過誘導(dǎo)腫瘤細(xì)胞的EMT發(fā)生而介導(dǎo)吉西他濱的耐藥,提示microRNA-21可能是乳腺癌逆轉(zhuǎn)耐藥的潛在治療靶點(diǎn)之一。

      [1] ZHANG J, WANG Z, HU X, et al. Cisplatin and gemcitabine as the first line therapy in metastatic triple negative breast cancer [J]. Int J Cancer, 2015, 136(1): 204-211.

      [2] SHANG Y, CAI X, FAN D. Roles of epithelial-mesenchymal transition in cancer drug resistance [J]. Curr Cancer Drug Targets, 2013, 13(9): 915-929.

      [3] MALLINI P, LENNARD T, KIRBY J, et al. Epithelial-tomesenchymal transition: what is the impact on breast cancer stem cells and drug resistance [J]. Cancer Treat Rev, 2014, 40(3): 341-348.

      [4] SINGH A, SETTLEMAN J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer [J]. Oncogene, 2010, 29(34): 4741-4751.

      [5] WANG Z, LI Y, AHMAD A, et al. Targeting miRNAs involved in cancer stem cell and EMT regulation: An emerging concept in overcoming drug resistance [J] . Drug Resist Updat, 2010, 13(4-5): 109-118.

      [6] D’AMATO N C, HOWE E N, RICHER J K. MicroRNA regulation of epithelial plasticity in cancer [J]. Cancer Lett, 2013, 341(1): 46-55.

      [7] YAN J, GUMIREDDY K, LI A, et al. Regulation of mesenchymal phenotype by MicroRNAs in cancer [J]. Curr Cancer Drug Targets, 2013, 13(9): 930-934.

      [8] SHI Z, ZHANG J, QIAN X, et al. AC1MMYR2, an inhibitor of dicer-mediated biogenesis of Oncomir miR-21, reverses epithelial-mesenchymal transition and suppresses tumor growth and progression [J]. Cancer Res, 2013, 73(17): 5519-5531.

      [9] HONG L, HAN Y, ZHANG Y, et al. MicroRNA-21: a therapeutic target for reversing drug resistance in cancer[J]. Expert Opin Ther Targets, 2013, 17(9): 1073-1080.

      [10] YANG X, LIN F, GUO Y, et al. Gemcitabine resistance in breast cancer cells regulated by PI3K/AKT-mediated cellular proliferation exerts negative feedback via the MEK/MAPK and mTOR pathways [J]. Onco Targets Ther, 2014, 7: 1033-1042.

      [11] BERGMAN A M, PINEDO H M, PETERS G J. Determinants of resistance to 2’, 2’-difluorodeoxycytidine (gemcitabine)[J]. Drug Resist Updat, 2002, 5(1): 19-33.

      [12] HAGMANN W, JESNOWSKI R, LOHR J M. Interdependence of gemcitabine treatment, transporter expression, and resistance in human pancreatic carcinoma cells [J]. Neoplasia, 2010, 12(9): 740-747.

      [13] ANDERSSON R, AHO U, NILSSON B I, et al. Gemcitabine chemoresistance in pancreatic cancer: molecular mechanisms and potential solutions [J] . Scand J Gastroenterol, 2009, 44(7): 782-786.

      [14] KIM M P, GALLICK G E. Gemcitabine resistance in pancreatic cancer: picking the key players [J] . Clin Cancer Res, 2008, 14(5): 1284-1285.

      [15] DAVIDSON J D, MA L, FLAGELLA M, et al. An increase in the expression of ribonucleotide reductase large subunit 1 is associated with gemcitabine resistance in non-small cell lung cancer cell lines [J] . Cancer Res, 2004, 64(11): 3761-3766.

      [16] SEBASTIANI V, RICCI F, RUBIO-VIQUEIRA B, et al.Immunohistochemical and genetic evaluation of deoxycytidine kinase in pancreatic cancer: relationship to molecular mechanisms of gemcitabine resistance and survival [J]. Clin Cancer Res, 2006, 12(8): 2492-2497.

      [17] RHA S Y, JEUNG H C, CHOI Y H, et al. An association between RRM1 haplotype and gemcitabine-induced neutropenia in breast cancer patients [J]. Oncologist, 2007, 12(6): 622-630.

      [18] HERNANDEZ-VARGAS H, RODRIGUEZ-PINILLA S M, JULIAN-TENDERO M, et al. Gene expression profiling of breast cancer cells in response to gemcitabine: NF-kappaB pathway activation as a potential mechanism of resistance[J]. Breast Cancer Res Treat, 2007, 102(2): 157-172.

      [19] XIA H, OOI L L, HUI K M. MicroRNA-216a/217-induced epithelial-mesenchymal transition targets PTEN and SMAD7 to promote drug resistance and recurrence of liver cancer[J]. Hepatology, 2013, 58(2): 629-641.

      [20] KITAMURA K, SEIKE M, OKANO T, et al. MiR-134/487b/655 cluster regulates TGF-beta-induced epithelialmesenchymal transition and drug resistance to gefitinib by targeting MAGI2 in lung adenocarcinoma cells [J]. Mol Cancer Ther, 2014, 13(2): 444-453.

      [21] PETROVIC N, MANDUSIC V, STANOJEVIC B, et al. The difference in miR-21 expression levels between invasive and non-invasive breast cancers emphasizes its role in breast cancer invasion [J]. Med Oncol, 2014, 31(3): 867-875.

      The underlying mechanism of microRNA-21 in gemcitabine resistant breast cancer cells

      WU Zhenhua, TAO Zhonghua, ZHANG Jian, XIE Jie, HU Xichun (Department of Medical Oncology, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China)

      HU Xichun E-mail: xchu2009@hotmail.com

      Background and purpose:Gemcitabine-based chemotherapy has been shown to have significant activity and favourable safety in metastatic breast cancer patients, but the effectiveness is limited due to drug resistance. MicroRNAs are a family of small non-coding RNA molecules, acting as oncogenes or tumor suppressors. Although various mechanisms of chemoresistance have been uncovered, the aberrant microRNA expression and its relationship with drug resistance of breast cancer are still unclear. This study explored the potential role and underlying mechanism of microRNA-21 in gemcitabine resistant breast cancer.Methods:MDA-MB-231 cells were continuously exposed to the increasing concentrations of gemcitabine to induce drug resistance to gemcitabine, which was 10 times more resistant. Then multiple methods were used including real-time PCR (RT-PCR), CCK-8, Western blot, transfection, wound healing and Transwell assay to observe the effect of microRNA-21 on epithelial-mesenchymal transition (EMT) and chemosensitivity.Results:The expression of microRNA-21 was up-regulated in gemcitabine resistant breast cancer cell line and inversely correlated with gemcitabine sensitivity. Manipulation of microRNA-21 status could change microRNA-21 level, and could result in corresponding changes in EMT status and drug sensitivity.Conclusion:MicroRNA-21 induces gemcitabine resistance possibly via EMT process in breast cancer.

      MicroRNA-21; Breast cancer; Gemcitabine resistance; Epithelial to mesenchymal transition

      10.3969/j.issn.1007-3969.2015.05.002

      R737.9

      A

      1007-3639(2015)05-0326-07

      2014-12-13

      2015-02-09)

      國家自然科學(xué)基金面上項(xiàng)目(81372846)。

      胡夕春 E-mail:xchu2009@hotmail.com

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