顧玉青 李占軍 張靜靜 高文濤 錢祝銀

·論著·

MicroRNA-200b在吉西他濱誘導的胰腺癌細胞株MiaPaCa-2上皮間質(zhì)轉化中的作用

顧玉青 李占軍 張靜靜 高文濤 錢祝銀

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

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

胰腺癌發(fā)現(xiàn)時多屬晚期，手術切除率低，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胞的上皮間質(zhì)轉化(epithelial-mesenchymal transition, EMT)相關[2-3]。EMT是指上皮細胞向間質(zhì)細胞表型轉化的過程。在EMT過程中，細胞表面上皮標志物E-鈣黏蛋白(E-cadherin)表達下調(diào)，間質(zhì)標志物波形蛋白(Vimentin)表達上調(diào)，導致上皮細胞失去細胞極性，胞間連接被破壞，胞內(nèi)肌動蛋白骨架重組[4-5]。而EMT在腫瘤的轉移和耐藥過程中起著重要作用[6]。微小RNA(microRNA，miR)在EMT過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]。應用靶基因預測軟件miRanda、TargetScan及TarBase發(fā)現(xiàn)miR-200b與Zeb1、Zeb2 mRNA 3′UTR區(qū)存在靶向配對關系。為此，本研究探討miR-200b在耐吉西他濱的胰腺癌MiaPaCa-2細胞發(fā)生EMT過程中所起的作用。

材料與方法

一、耐吉西他濱的胰腺癌MiaPaCa-2細胞株的誘導及miR-200b轉染

人胰腺癌細胞株MiaPaCa-2由本實驗室保存，常規(guī)傳代培養(yǎng)。應用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細胞48 h，獲得50%細胞生長抑制時的藥物濃度(IC50)，以該濃度誘導的細胞為耐藥細胞株。

取對數(shù)生長期的MiaPaCa-2細胞接種于6孔板中，培養(yǎng)24 h待細胞融合度在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)轉染細胞，以轉染無意義小分子片段作為陰性對照。再用IC50的吉西他濱處理轉染細胞24 h，獲得轉染miR-200b的耐藥細胞株及陰性對照的耐藥細胞株。

二、細胞侵襲實驗

Transwell小室膜上層預鋪1 mg/ml的Matrigel。收集對數(shù)生長期親本MiaPaCa-2細胞、耐藥MiaPaCa-2細胞、陰性對照的耐藥MiaPaCa-2細胞和轉染miR-200b的耐藥MiaPaCa-2細胞，用無血清細胞培養(yǎng)基DMEM重懸各種細胞，上室加1×105個細胞，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，應用0.1%結晶紫染色，倒置顯微鏡下觀察。隨機取5個100倍視野，計算穿膜細胞數(shù)，取均值。實驗重復3次。

三、蛋白質(zhì)印跡法

收集對數(shù)生長期MiaPaCa-2細胞、耐藥MiaPaCa-2細胞、陰性對照的耐藥MiaPaCa-2細胞和轉染miR-200b的MiaPaCa-2細胞，采用蛋白裂解液RIPA提取細胞總蛋白。常規(guī)行蛋白質(zhì)印跡法檢測EMT相關蛋白E-cadherin、Vimentin及miR-200b的靶向蛋白Zeb1、Zeb2的表達，以GAPDH為內(nèi)參。兔抗人Zeb1抗體，鼠抗人Zeb2、E-cadherin、Vimentin抗體均購自Abcam公司，ECL發(fā)光試劑盒購自碧云天公司。

四、實時定量PCR法

收集對數(shù)生長期MiaPaCa-2細胞及耐藥MiaPaCa-2細胞，采用Trizol法提取細胞總RNA。先將miR-200b特異性反轉錄引物逆轉成cDNA，再行PCR擴增，以U6為內(nèi)參。PCR反應條件：95℃ 10 min， 95℃ 15 s、60℃ 60 s，40個循環(huán)。TaqMan熒光探針購自Roche公司。目的基因mRNA表達量依據(jù)公式2-△△Ct進行計算，實驗重復3次，取均值。

五、統(tǒng)計學處理

結 果

一、吉西他濱誘導后細胞形態(tài)變化

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

二、耐藥MiaPaCa-2細胞EMT相關蛋白的表達

耐藥MiaPaCa-2細胞的E-cadherin表達下調(diào)，為親本細胞的(0.47±0.05)倍；Vimentin、Zeb1、Zeb2表達上調(diào)，分別為親本細胞的(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：存活細胞曲線)

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

三、耐藥MiaPaCa-2細胞miR-200b的表達

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

四、轉染miR-200b的耐藥MiaPaCa-2細胞形態(tài)及侵襲能力的變化

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

圖3MiaPaCa-2細胞(a)、耐藥MiaPaCa-2細胞(b)、陰性對照的耐藥MiaPaCa-2細胞(c)及轉染miR-200b的耐藥MiaPaCa-2細胞(d)的形態(tài)變化(左)及穿膜細胞(右 ×100)

五、轉染miR-200b的耐藥MiaPaCa-2細胞Zeb1、Zeb2蛋白的表達

轉染miR-200b的耐藥MiaPaCa-2細胞Zeb1、Zeb2蛋白表達較陰性對照的耐藥MiaPaCa-2細胞顯著下調(diào)，分別為(0.36±0.07)、(0.08±0.01)倍(P值均<0.01)，陰性對照的耐藥MiaPaCa-2細胞的表達與耐藥MiaPaCa-2細胞的表達差異無統(tǒng)計學意義(圖4)。

圖4耐藥MiaPaCa-2細胞(1)、陰性對照的耐藥MiaPaCa-2細胞(2)及轉染miR-200b的耐藥MiaPaCa-2細胞(3)的Zeb1、Zeb2蛋白表達

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，近年來研究發(fā)現(xiàn)腫瘤對吉西他濱耐藥可能與EMT相關[8]。本研究應用吉西他濱誘導胰腺癌MiaPaCa-2細胞，結果顯示吉西他濱誘導后MiaPaCa-2細胞的E-cadherin表達下調(diào)，Vimentin表達上調(diào)，表明MiaPaCa-2細胞出現(xiàn)了EMT現(xiàn)象。

已有研究證實，miR-200b參與調(diào)控腫瘤細胞的侵襲和遷移能力[9]。本研究結果顯示，吉西他濱誘導后MiaPaCa-2細胞的miR-200b表達明顯下調(diào)，而穿膜細胞數(shù)明顯增加，提示耐藥的MiaPaCa-2細胞更具侵襲力，而miR-200b可能參與此過程。將miR-200b轉染MiaPaCa-2細胞后再用吉西他濱誘導，則轉染miR-200b的耐藥MiaPaCa-2細胞的穿膜細胞數(shù)明顯減少，Zeb1、Zeb2蛋白表達下調(diào)。文獻報道，Zeb1、Zeb2蛋白能夠下調(diào)E-cadherin或者直接調(diào)控EMT而參與腫瘤細胞侵襲和遷移[10]。本研究結果表明，轉染miR-200b的耐藥MiaPaCa-2細胞的EMT過程放緩，細胞的侵襲力降低，提示miR-200b可能通過靶向調(diào)節(jié)Zeb1、Zeb2蛋白而參與EMT過程，降低細胞的侵襲力，這一結論為miR-200b的靶向治療應用提供了實驗依據(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)科大學第一附屬醫(yī)院膽胰外科

錢祝銀，Email：qianzhusilver@163.com