顧玉青 李占軍 張靜靜 高文濤 錢祝銀

·論著·

MicroRNA-200b在吉西他濱誘導(dǎo)的胰腺癌細(xì)胞株MiaPaCa-2上皮間質(zhì)轉(zhuǎn)化中的作用

顧玉青 李占軍 張靜靜 高文濤 錢祝銀

目的探討MicroRNA-200b(miR-200b)在吉西他濱誘導(dǎo)的胰腺癌MiaPaCa-2細(xì)胞上皮間質(zhì)轉(zhuǎn)化(EMT)過(guò)程中的作用。方法應(yīng)用不同濃度的吉西他濱誘導(dǎo)MiaPaCa-2細(xì)胞，選擇50%細(xì)胞生長(zhǎng)抑制時(shí)的藥物濃度(IC50)，獲取耐藥MiaPaCa-2細(xì)胞。采用脂質(zhì)體法將miR-200b和無(wú)意義小分子片段(陰性對(duì)照)分別轉(zhuǎn)染MiaPaCa-2細(xì)胞，再用IC50的吉西他濱誘導(dǎo)細(xì)胞，獲取轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞及陰性對(duì)照的耐藥MiaPaCa-2細(xì)胞。倒置顯微鏡下觀察細(xì)胞形態(tài)變化；Transwell小室測(cè)定細(xì)胞侵襲能力；實(shí)時(shí)定量PCR檢測(cè)細(xì)胞miR-200b表達(dá)；蛋白質(zhì)印跡法檢測(cè)細(xì)胞E-cadherin、Vimentin、Zeb1、Zeb2蛋白表達(dá)。結(jié)果吉西他濱處理后細(xì)胞體積逐漸縮小，呈紡錘樣，細(xì)胞間連接減少，偽足增多，呈現(xiàn)間質(zhì)細(xì)胞特征。耐藥MiaPaCa-2細(xì)胞的穿膜數(shù)從親本細(xì)胞的(26±3)個(gè)上升至(85±6)個(gè)，Vimentin、Zeb1、Zeb2表達(dá)分別上升至親本細(xì)胞的(1.87±0.17)、(2.57±0.21)、(5.24±0.83)倍，miR-200b表達(dá)下降至親本細(xì)胞的(0.36±0.01)倍，E-cadherin表達(dá)下降至親本細(xì)胞的(0.47±0.05)倍。而轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞的穿膜數(shù)下降至(42±4)個(gè)，Zeb1、Zeb2表達(dá)下降至陰性對(duì)照的耐藥MiaPaCa-2細(xì)胞的(0.36±0.07)、(0.08±0.01)倍。結(jié)論吉西他濱誘導(dǎo)胰腺癌MiaPaCa-2細(xì)胞過(guò)程中細(xì)胞出現(xiàn)EMT，其機(jī)制可能與miR-200b表達(dá)下調(diào)有關(guān)。

胰腺腫瘤； 微RNAs； miR-200b； 吉西他濱； 上皮間質(zhì)轉(zhuǎn)化

胰腺癌發(fā)現(xiàn)時(shí)多屬晚期，手術(shù)切除率低，化療是其重要的輔助治療手段[1]。近期有研究發(fā)現(xiàn)，包括放療、化療在內(nèi)的抗腫瘤治療可能與腫瘤細(xì)胞的上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition, EMT)相關(guān)[2-3]。EMT是指上皮細(xì)胞向間質(zhì)細(xì)胞表型轉(zhuǎn)化的過(guò)程。在EMT過(guò)程中，細(xì)胞表面上皮標(biāo)志物E-鈣黏蛋白(E-cadherin)表達(dá)下調(diào)，間質(zhì)標(biāo)志物波形蛋白(Vimentin)表達(dá)上調(diào)，導(dǎo)致上皮細(xì)胞失去細(xì)胞極性，胞間連接被破壞，胞內(nèi)肌動(dòng)蛋白骨架重組[4-5]。而EMT在腫瘤的轉(zhuǎn)移和耐藥過(guò)程中起著重要作用[6]。微小RNA(microRNA，miR)在EMT過(guò)程中有著舉足輕重的作用，其中miR-200家族是最重要的一類[7]。應(yīng)用靶基因預(yù)測(cè)軟件miRanda、TargetScan及TarBase發(fā)現(xiàn)miR-200b與Zeb1、Zeb2 mRNA 3′UTR區(qū)存在靶向配對(duì)關(guān)系。為此，本研究探討miR-200b在耐吉西他濱的胰腺癌MiaPaCa-2細(xì)胞發(fā)生EMT過(guò)程中所起的作用。

材料與方法

一、耐吉西他濱的胰腺癌MiaPaCa-2細(xì)胞株的誘導(dǎo)及miR-200b轉(zhuǎn)染

人胰腺癌細(xì)胞株MiaPaCa-2由本實(shí)驗(yàn)室保存，常規(guī)傳代培養(yǎng)。應(yīng)用0、0.01、0.02、0.04、0.08、0.16、0.32、0.64、1、10、100 μmol/L吉西他濱處理MiaPaCa-2細(xì)胞48 h，獲得50%細(xì)胞生長(zhǎng)抑制時(shí)的藥物濃度(IC50)，以該濃度誘導(dǎo)的細(xì)胞為耐藥細(xì)胞株。

取對(duì)數(shù)生長(zhǎng)期的MiaPaCa-2細(xì)胞接種于6孔板中，培養(yǎng)24 h待細(xì)胞融合度在60%左右，采用脂質(zhì)體Lipofectamine 2000將miR-200b模擬物(上海吉瑪公司合成，序列為UAAUACUGCCUGGUAAUG-AUGA)轉(zhuǎn)染細(xì)胞，以轉(zhuǎn)染無(wú)意義小分子片段作為陰性對(duì)照。再用IC50的吉西他濱處理轉(zhuǎn)染細(xì)胞24 h，獲得轉(zhuǎn)染miR-200b的耐藥細(xì)胞株及陰性對(duì)照的耐藥細(xì)胞株。

二、細(xì)胞侵襲實(shí)驗(yàn)

Transwell小室膜上層預(yù)鋪1 mg/ml的Matrigel。收集對(duì)數(shù)生長(zhǎng)期親本MiaPaCa-2細(xì)胞、耐藥MiaPaCa-2細(xì)胞、陰性對(duì)照的耐藥MiaPaCa-2細(xì)胞和轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞，用無(wú)血清細(xì)胞培養(yǎng)基DMEM重懸各種細(xì)胞，上室加1×105個(gè)細(xì)胞，下室加含10%胎牛血清的DMEM培養(yǎng)基0.5 ml，常規(guī)培養(yǎng)24 h，應(yīng)用0.1%結(jié)晶紫染色，倒置顯微鏡下觀察。隨機(jī)取5個(gè)100倍視野，計(jì)算穿膜細(xì)胞數(shù)，取均值。實(shí)驗(yàn)重復(fù)3次。

三、蛋白質(zhì)印跡法

收集對(duì)數(shù)生長(zhǎng)期MiaPaCa-2細(xì)胞、耐藥MiaPaCa-2細(xì)胞、陰性對(duì)照的耐藥MiaPaCa-2細(xì)胞和轉(zhuǎn)染miR-200b的MiaPaCa-2細(xì)胞，采用蛋白裂解液RIPA提取細(xì)胞總蛋白。常規(guī)行蛋白質(zhì)印跡法檢測(cè)EMT相關(guān)蛋白E-cadherin、Vimentin及miR-200b的靶向蛋白Zeb1、Zeb2的表達(dá)，以GAPDH為內(nèi)參。兔抗人Zeb1抗體，鼠抗人Zeb2、E-cadherin、Vimentin抗體均購(gòu)自Abcam公司，ECL發(fā)光試劑盒購(gòu)自碧云天公司。

四、實(shí)時(shí)定量PCR法

收集對(duì)數(shù)生長(zhǎng)期MiaPaCa-2細(xì)胞及耐藥MiaPaCa-2細(xì)胞，采用Trizol法提取細(xì)胞總RNA。先將miR-200b特異性反轉(zhuǎn)錄引物逆轉(zhuǎn)成cDNA，再行PCR擴(kuò)增，以U6為內(nèi)參。PCR反應(yīng)條件：95℃ 10 min， 95℃ 15 s、60℃ 60 s，40個(gè)循環(huán)。TaqMan熒光探針購(gòu)自Roche公司。目的基因mRNA表達(dá)量依據(jù)公式2-△△Ct進(jìn)行計(jì)算，實(shí)驗(yàn)重復(fù)3次，取均值。

五、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、吉西他濱誘導(dǎo)后細(xì)胞形態(tài)變化

親本MiaPaCa-2細(xì)胞體積略大，呈多邊形，團(tuán)簇狀生長(zhǎng)；吉西他濱處理的細(xì)胞隨藥物增加細(xì)胞體積逐漸縮小，呈紡錘樣，且細(xì)胞間連接減少，偽足增多，細(xì)胞排列無(wú)規(guī)則，呈現(xiàn)間質(zhì)細(xì)胞特征(圖1)，經(jīng)活細(xì)胞計(jì)數(shù)，吉西他濱的IC50為62 nmol/L。

二、耐藥MiaPaCa-2細(xì)胞EMT相關(guān)蛋白的表達(dá)

耐藥MiaPaCa-2細(xì)胞的E-cadherin表達(dá)下調(diào)，為親本細(xì)胞的(0.47±0.05)倍；Vimentin、Zeb1、Zeb2表達(dá)上調(diào)，分別為親本細(xì)胞的(1.87±0.17)、(2.57±0.21)、(5.24±0.83)倍(P值均<0.05，圖2)。

圖1吉西他濱處理后的MiaPaCa-2細(xì)胞(a～k：0、0.01、0.02、0.04、0.08、0.16、0.32、0.64、1、10、100 μmol/L吉西他濱，倒置顯微 ×200，l：存活細(xì)胞曲線)

圖2MiaPaCa-2細(xì)胞(1)及耐藥MiaPaCa-2細(xì)胞(2)的Zeb1、Zeb2、E-cadherin及Vimentin蛋白表達(dá)

三、耐藥MiaPaCa-2細(xì)胞miR-200b的表達(dá)

耐藥MiaPaCa-2細(xì)胞的miR-200b表達(dá)量下降至親本MiaPaCa-2細(xì)胞的(0.36±0.01)倍(3.93比5.41，P<0.01)。

四、轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞形態(tài)及侵襲能力的變化

MiaPaCa-2細(xì)胞、耐藥MiaPaCa-2細(xì)胞、陰性對(duì)照的耐藥MiaPaCa-2細(xì)胞及轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞的穿膜細(xì)胞數(shù)分別為(26±3)、(85±6)、(81±7)、(42±4)個(gè)。耐藥MiaPaCa-2細(xì)胞的穿膜數(shù)較親本MiaPaCa-2細(xì)胞顯著增多，轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞的穿膜數(shù)較耐藥MiaPaCa-2細(xì)胞及陰性對(duì)照的耐藥MiaPaCa-2細(xì)胞顯著減少(P值均<0.01)，但仍較親本MiaPaCa-2細(xì)胞顯著增加(P<0.05，圖3)。

圖3MiaPaCa-2細(xì)胞(a)、耐藥MiaPaCa-2細(xì)胞(b)、陰性對(duì)照的耐藥MiaPaCa-2細(xì)胞(c)及轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞(d)的形態(tài)變化(左)及穿膜細(xì)胞(右 ×100)

五、轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞Zeb1、Zeb2蛋白的表達(dá)

轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞Zeb1、Zeb2蛋白表達(dá)較陰性對(duì)照的耐藥MiaPaCa-2細(xì)胞顯著下調(diào)，分別為(0.36±0.07)、(0.08±0.01)倍(P值均<0.01)，陰性對(duì)照的耐藥MiaPaCa-2細(xì)胞的表達(dá)與耐藥MiaPaCa-2細(xì)胞的表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(圖4)。

圖4耐藥MiaPaCa-2細(xì)胞(1)、陰性對(duì)照的耐藥MiaPaCa-2細(xì)胞(2)及轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞(3)的Zeb1、Zeb2蛋白表達(dá)

討 論

吉西他濱已經(jīng)成為晚期胰腺癌的一線化療藥物，但療效仍不理想，近年來(lái)研究發(fā)現(xiàn)腫瘤對(duì)吉西他濱耐藥可能與EMT相關(guān)[8]。本研究應(yīng)用吉西他濱誘導(dǎo)胰腺癌MiaPaCa-2細(xì)胞，結(jié)果顯示吉西他濱誘導(dǎo)后MiaPaCa-2細(xì)胞的E-cadherin表達(dá)下調(diào)，Vimentin表達(dá)上調(diào)，表明MiaPaCa-2細(xì)胞出現(xiàn)了EMT現(xiàn)象。

已有研究證實(shí)，miR-200b參與調(diào)控腫瘤細(xì)胞的侵襲和遷移能力[9]。本研究結(jié)果顯示，吉西他濱誘導(dǎo)后MiaPaCa-2細(xì)胞的miR-200b表達(dá)明顯下調(diào)，而穿膜細(xì)胞數(shù)明顯增加，提示耐藥的MiaPaCa-2細(xì)胞更具侵襲力，而miR-200b可能參與此過(guò)程。將miR-200b轉(zhuǎn)染MiaPaCa-2細(xì)胞后再用吉西他濱誘導(dǎo)，則轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞的穿膜細(xì)胞數(shù)明顯減少，Zeb1、Zeb2蛋白表達(dá)下調(diào)。文獻(xiàn)報(bào)道，Zeb1、Zeb2蛋白能夠下調(diào)E-cadherin或者直接調(diào)控EMT而參與腫瘤細(xì)胞侵襲和遷移[10]。本研究結(jié)果表明，轉(zhuǎn)染miR-200b的耐藥MiaPaCa-2細(xì)胞的EMT過(guò)程放緩，細(xì)胞的侵襲力降低，提示miR-200b可能通過(guò)靶向調(diào)節(jié)Zeb1、Zeb2蛋白而參與EMT過(guò)程，降低細(xì)胞的侵襲力，這一結(jié)論為miR-200b的靶向治療應(yīng)用提供了實(shí)驗(yàn)依據(jù)。

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EffectofmiR-200bongemcitabineinducedepithelialmesenchymaltransitioninpancreaticcancercelllineMiaPaCa-2

GUYu-qing,LIZhan-jun,ZHANGJing-jing,GAOWen-tao,QIANZhu-yin.

DepartmentofGeneralSurgery,FirstAffiliatedHospital,NanjingMedicalUniversity,Nanjing210029,China

Correspondingauthor:QIANZhu-yin,Email:qianzhusilver@163.com

ObjectiveTo investigate the role of miR-200b on gemcitabine induced epithelial-mesenchymal transition (EMT) in pancreatic cancer cell line MiaPaCa-2.MethodsDifferent concentrations of gemcitabine were used to induce MiaPaCa-2, and the concentration of 50% cell proliferation inhibited (IC50) was applied to obtain drug-resistant MiaPaCa-2 cells. MiR-200b or nonsense small molecular fragments (negative control,NC) was transfected into MiaPaCa-2 cells by liposomes, then gemcitabine of IC50was used to induce cells to obtain drug-resistant MiaPaCa-2 cells transfected with miR-200b or NC. The morphological characteristics of MiaPaCa-2 cells were observed by inverted microscope. Invasion of cells were detected by transwell chamber. The expression of miR-200b was measured by using real-time PCR. The expressions of E-cadherin, Vimentin, Zeb1, Zeb2 proteins were determined by Western blot.ResultsAfter gemcitabine treatment, the cells′ size gradually diminished, intercellular junctions decreased, pseudopodium increased, which presented the characteristics of mesenchymal morphology. The invaded cell number increased from (26±3) to (85±6), and the expression of Vimentin Zeb1, Zeb2 was increased to (1.87±0.17), (2.57±0.21), (5.24±0.83) folds of the parent cells. The expression of miR-200b was decreased to (0.36±0.01) folds of the parent cells, and the expression of E-cadherin was decreased to 0.47±0.05 folds of the parent cells, while the invaded cell number of drug-resistant MiaPaCa-2 transfected with miR-200b was decreased to (42±4), and the expression of Zeb1, Zeb2 was decreased to (0.36±0.07), (0.08±0.01) folds of drug-resistant MiaPaCa-2 transfected with NC.ConclusionsThe occurrence of EMT is observed in pancreatic cancer cell line MiaPaCa-2 during gemcitabine induction, and miR-200b down-regulation may be a possible mechanism.

Pancreatic neoplasms; MicroRNAs; miR-200b; Gemcitabine; Epithelial-mesenchymal transition

2013-02-28)

(本文編輯：呂芳萍)

10.3760/cma.j.issn.1674-1935.2013.04.010

210029 江蘇南京，南京醫(yī)科大學(xué)第一附屬醫(yī)院膽胰外科

錢祝銀，Email：qianzhusilver@163.com