低氧培養(yǎng)對胰腺癌CFPAC-1細(xì)胞miR-301a表達(dá)的影響

朱麟　張坤東　夏翔　黃陳　裘正軍

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·論著·

低氧培養(yǎng)對胰腺癌CFPAC-1細(xì)胞miR-301a表達(dá)的影響

朱麟張坤東夏翔黃陳裘正軍

200080上海，上海交通大學(xué)附屬第一人民醫(yī)院普外科，上海市胰腺疾病重點實驗室，上海交通大學(xué)胰腺癌診治中心

【摘要】目的觀察低氧培養(yǎng)胰腺癌CFPAC-1細(xì)胞后miR-301a表達(dá)的變化，探討miR-301a的可能作用機制。方法在1% O2環(huán)境中培養(yǎng)胰腺癌細(xì)胞CFPAC-1，相差顯微鏡下觀察細(xì)胞形態(tài)變化。蛋白質(zhì)印跡法檢測細(xì)胞低氧誘導(dǎo)因子HIF-1α、miR-301a及其靶基因FOXF2，上皮型標(biāo)志物E-cadherin、ZO-1、β-catenin，上皮間質(zhì)轉(zhuǎn)化(EMT)標(biāo)志物Vimentin、N-cadherin、Fibronectin的表達(dá)。構(gòu)建miR-301a慢病毒表達(dá)載體，建立穩(wěn)定轉(zhuǎn)染miR-301a的細(xì)胞株，以轉(zhuǎn)染與miR-301a不匹配(miR-301a-NC)的細(xì)胞作為對照組，觀察細(xì)胞形態(tài)變化，檢測FOXF2、上皮型及EMT標(biāo)志物表達(dá)，劃痕實驗和Transwell小室實驗檢測細(xì)胞遷移、侵襲能力。結(jié)果CFPAC-1細(xì)胞在低氧環(huán)境培養(yǎng)后細(xì)胞外形不規(guī)則、多呈棱形、偽足較多，細(xì)胞間連接松散，向間質(zhì)細(xì)胞形態(tài)改變。以常氧培養(yǎng)細(xì)胞的表達(dá)量為1，低氧培養(yǎng)4 d后細(xì)胞miR-301a表達(dá)量為2.82±0.01，HIF-1α為2.17±0.36，Vimentin、N-cadherin、Fibronectin分別為5.48±0.16、1.16±0.03、1.30±0.03，均顯著上調(diào)；FOXF2、E-cadherin、ZO-1、β-catenin分別為0.45±0.05、0.61±0.05、0.45±0.02、0.37±0.02，均顯著下調(diào)，差異均有統(tǒng)計學(xué)意義(P值均<0.01)。以轉(zhuǎn)染miR-301a-NC細(xì)胞的蛋白表達(dá)量為1，轉(zhuǎn)染miR-301a細(xì)胞的miR-301a、Fibronectin、Vimentin、N-cadherin表達(dá)量分別為7.09±0.42、1.56±0.10、1.24±0.05、1.37±0.01，表達(dá)均顯著上調(diào)；FOXF2、ZO-1、E-cadherin、β-catenin表達(dá)量分別為0.33±0.03、0.71±0.02、0.67±0.06、0.47±0.03，表達(dá)均顯著下調(diào)，差異均有統(tǒng)計學(xué)意義(P值均<0.01)。轉(zhuǎn)染miR-301a-NC組、穩(wěn)轉(zhuǎn)miR-301a組CFPAC-1細(xì)胞18 h的覆蓋劃痕區(qū)面積分別為(54.68±4.31)%、(94.82±2.18)%；24 h的穿膜細(xì)胞數(shù)分別為(25±6)、(95±11)/100倍視野，穩(wěn)轉(zhuǎn)miR-301a組顯著高于轉(zhuǎn)染miR-301a-NC組，差異均有統(tǒng)計學(xué)意義(P<0.05或<0.01)。結(jié)論低氧培養(yǎng)后CFPAC-1細(xì)胞出現(xiàn)EMT改變，miR-301a表達(dá)上調(diào)。其機制可能是miR-301a通過抑制FOXF2表達(dá)從而促使胰腺癌細(xì)胞EMT。

【關(guān)鍵詞】胰腺腫瘤；缺氧；上皮間質(zhì)轉(zhuǎn)化；微RNAs

Fund Program: National Natural Science Foundation of China (81372640)

實體腫瘤中存在缺血壞死現(xiàn)象，提示腫瘤處于低氧狀態(tài)，而低氧微環(huán)境是誘導(dǎo)胰腺癌高侵襲性的重要因素[1]。上皮間質(zhì)轉(zhuǎn)化(EMT)是上皮細(xì)胞來源的腫瘤細(xì)胞侵襲轉(zhuǎn)移的一個重要步驟。Cheng等[2]的研究證實，缺氧可誘導(dǎo)HIF-1α的表達(dá)從而導(dǎo)致胰腺癌細(xì)胞發(fā)生EMT，促進(jìn)胰腺癌的侵襲轉(zhuǎn)移。近年來很多研究證實，miRNA參與腫瘤細(xì)胞EMT及轉(zhuǎn)移過程[3-7]。miR-301a在乳腺癌[8]、肝癌[9]、結(jié)直腸癌[10]、胃癌[11-12]以及胰腺癌[13]中表達(dá)升高，在腫瘤增殖、凋亡、血管生成和侵襲轉(zhuǎn)移中發(fā)揮重要作用，然而miR-301a與胰腺癌進(jìn)展的相關(guān)性尚不明確，其在胰腺癌EMT中的作用尚未見報道。本研究初步探討miR-301a在低氧誘導(dǎo)胰腺癌細(xì)胞EMT中的作用及其相關(guān)機制。

材料與方法

一、細(xì)胞培養(yǎng)

人胰腺癌細(xì)胞系CFPAC-1購自美國ATCC細(xì)胞庫。細(xì)胞復(fù)蘇后應(yīng)用含10%胎牛血清(GIBCO)的IMDM培養(yǎng)液，置于37℃、5% CO2、飽和濕度的常氧培養(yǎng)箱及含1% O2的低氧培養(yǎng)箱中分別培養(yǎng)2、4 d，在相差顯微鏡下觀察細(xì)胞形態(tài)。

二、實時PCR

三、蛋白質(zhì)印跡法

用SDS裂解液提取細(xì)胞蛋白，煮沸變性后常規(guī)行蛋白質(zhì)印跡法檢測細(xì)胞低氧誘導(dǎo)因子HIF-1α、miR-301a及其靶基因FOXF2，上皮型標(biāo)志物E-cadherin、ZO-1、β-catenin，上皮間質(zhì)轉(zhuǎn)化標(biāo)志物Vimentin、Fibronection、N-cadherin蛋白表達(dá)，以β-actin為內(nèi)參?？笶-cadherin、ZO-1、β-catenin、Fibronection、N-cadherin一抗均購自美國Cell Signaling Technology公司，抗FOXF2一抗購自abcam公司，抗HIF-1α、Vimentin一抗購自BD Biosciences公司。最后ECL增強發(fā)光，X線曝光。應(yīng)用Quantity One軟件掃描，以目的條帶與內(nèi)參條帶灰度值比表示蛋白相對表達(dá)量。

四、miR-301a穩(wěn)轉(zhuǎn)細(xì)胞株建立

應(yīng)用Primer 5軟件設(shè)計miR-301a擴(kuò)增引物，正義端加EcoRI酶切序列，反義端加BanHI酶切序列。正義序列5′-CCGGAATTCCTATTAAGGCTTAGGGC-ATA-3′；反義序列5′-CGCGGATTCCTCATTTAGACAAACCATAA-3′，擴(kuò)增產(chǎn)物581 bp。經(jīng)限制性內(nèi)切酶EcoRI、BamHI雙酶切后，用T4連接酶將酶切產(chǎn)物與線性化載體pLV-mCherry連接。按說明書操作。

采用脂質(zhì)體2000(Life Technologies公司)將LV-miR-301a-mCherry和包裝質(zhì)粒pMDG、pCMV△8.9共轉(zhuǎn)染293T細(xì)胞，培養(yǎng)48 h后收取病毒上清并濃縮。以與miR-301a不匹配(miR-301a-NC)的慢病毒表達(dá)載體pLV-miR-301a-NC-mCherry作為陰性對照組。將濃縮后的病毒上清加入含F(xiàn)BS的完全培養(yǎng)基中，感染處于對數(shù)生長期的CFPAC-1細(xì)胞48 h，應(yīng)用嘌呤霉素篩選miR-301a穩(wěn)定轉(zhuǎn)染的細(xì)胞。

五、劃痕實驗

收集穩(wěn)轉(zhuǎn)miR-301a的CFPAC-1細(xì)胞接種于6孔板，每孔約5×105個細(xì)胞，培養(yǎng)至細(xì)胞鋪滿后用槍頭垂直劃線，PBS洗滌3次去除劃下的細(xì)胞，加入培養(yǎng)液分別繼續(xù)培養(yǎng)12、18 h，以轉(zhuǎn)染miR-301a-NC的細(xì)胞作為對照組。觀察細(xì)胞遷移覆蓋劃痕區(qū)的情況。實驗重復(fù)3次。

土壤污染防治法規(guī)定，各類涉及土地利用的規(guī)劃和可能造成土壤污染的建設(shè)項目，應(yīng)當(dāng)依法進(jìn)行環(huán)境影響評價。各級人民政府生態(tài)環(huán)境、自然資源主管部門依法加強對礦產(chǎn)資源開發(fā)區(qū)域土壤污染防治的監(jiān)督管理，嚴(yán)控可能造成土壤污染的重點污染物排放。

六、Transwell實驗

收集穩(wěn)轉(zhuǎn)miR-301a的CFPAC-1細(xì)胞，用無血清培養(yǎng)基重懸，上室加入200 μl細(xì)胞懸液(5×104個細(xì)胞)，下室加入含10%血清的培養(yǎng)基700 μl，培養(yǎng)24 h后，用棉簽輕輕擦去上室未穿膜細(xì)胞，甲醇固定15 min，結(jié)晶紫染色3 h，100倍顯微鏡下計算4個視野的穿膜細(xì)胞數(shù)，取均值。以轉(zhuǎn)染miR-301a-NC的細(xì)胞作為對照組。

七、統(tǒng)計學(xué)處理

結(jié)果

一、低氧培養(yǎng)下CFPAC-1細(xì)胞的形態(tài)改變

常規(guī)培養(yǎng)的對照組細(xì)胞外形較規(guī)則、偽足較少、呈圓形或橢圓形，細(xì)胞間連接緊密(圖1A)；低氧培養(yǎng)后細(xì)胞外形不規(guī)則、多呈棱形、偽足較多，細(xì)胞間連接松散(圖1B)，提示細(xì)胞由上皮細(xì)胞形態(tài)向間質(zhì)細(xì)胞形態(tài)改變。

圖1　常氧培養(yǎng)(1A)與低氧培養(yǎng)組(1B)CFPAC-1細(xì)胞形態(tài)觀察(×100)

二、低氧培養(yǎng)后CFPAC-1細(xì)胞相關(guān)蛋白表達(dá)的變化(圖2)

低氧培養(yǎng)后2、4 d，細(xì)胞miR-301a表達(dá)為常規(guī)培養(yǎng)細(xì)胞的(2.71±0.04)、(2.82±0.01)倍，差異有統(tǒng)計學(xué)意義(t值分別為14.74、28.72，P值均<0.01)。以常氧培養(yǎng)細(xì)胞的蛋白表達(dá)量為1，低氧培養(yǎng)后2、4 d時細(xì)胞HIF-1α相對表達(dá)量為4.11±0.60、2.17±0.36，差異有統(tǒng)計學(xué)意義(t值分別為13.02、8.28，P值均<0.01)；FOXF2為0.40±0.04、0.45±0.05(t值分別為27.83、19.58)，E-cadherin為0.50±0.01、0.61±0.05(t值分別為76.86、13.79)，ZO-1為0.46±0.03、0.45±0.02(t值分別為36.17、45.42)，β-catenin為0.40±0.02、0.37±0.02(t值分別為46.28、45.13)，均顯著下調(diào)；Vimentin為1.73±0.09、5.48±0.16(t值分別為8.52、28.47)，N-cadherin為1.04±0.02、1.16±0.03(t值分別為1.83、5.41)，F(xiàn)ibronectin為1.20±0.04、1.30±0.03(t值分別為5.35、8.46)，均顯著上調(diào)。除低氧培養(yǎng)2 d的N-cadherin外，兩組間差異均有統(tǒng)計學(xué)意義(P值均<0.01)。

三、穩(wěn)轉(zhuǎn)miR-301a的CFPAC-1細(xì)胞

常氧培養(yǎng)下轉(zhuǎn)染miR-301a-NC細(xì)胞呈上皮細(xì)胞形態(tài)(圖3A)，轉(zhuǎn)染miR-301a細(xì)胞呈間質(zhì)細(xì)胞形態(tài)改變(圖3B)。

圖2　常氧(1)及低氧組2、4 d(2、3)CFPAC-1細(xì)胞相關(guān)蛋白的表達(dá)

圖3　轉(zhuǎn)染miR-301a-NC組(3A)及穩(wěn)轉(zhuǎn)miR-301a組(3B)CFPAC-1細(xì)胞的形態(tài)變化(×200倍)

轉(zhuǎn)染miR-301a細(xì)胞的miR-301a表達(dá)量為轉(zhuǎn)染miR-301a-NC細(xì)胞的(7.09±0.42)倍(t=14.47，P<0.01)。以轉(zhuǎn)染miR-301a-NC細(xì)胞的蛋白表達(dá)量為1，轉(zhuǎn)染miR-301a細(xì)胞的FOXF2、ZO-1、E-cadherin、β-catenin相對表達(dá)量分別為0.33±0.03、0.71±0.02、0.67±0.06、0.47±0.03，均顯著下調(diào)(t值分別為22.23、11.9、5.18、16.28)；Fibronectin、Vimentin、N-cadherin相對表達(dá)量分別為1.56±0.10、1.24±0.05、1.37±0.01，均顯著上調(diào)(t值分別為4.99、4.73、25.59)，兩組差異均有統(tǒng)計學(xué)意義(P值均<0.01，圖4)。

四、穩(wěn)轉(zhuǎn)細(xì)胞遷移能力的變化

轉(zhuǎn)染miR-301a-NC組、穩(wěn)轉(zhuǎn)miR-301a組CFPAC-1細(xì)胞0、12、18 h的遷移狀態(tài)如圖5所示。轉(zhuǎn)染miR-301a-NC組12、18 h時細(xì)胞覆蓋劃痕區(qū)面積為(28.26±4.55)%、(54.68±4.31)%；穩(wěn)轉(zhuǎn)miR-301a組的覆蓋率為(60.97±4.72)%、(94.82±2.18)%，穩(wěn)轉(zhuǎn)miR-301a組細(xì)胞遷移能力顯著高于轉(zhuǎn)染miR-301a-NC組，差異有統(tǒng)計學(xué)意義(t值分別為7.30、7.27，P值均<0.01)。

圖4　轉(zhuǎn)染miR-301a-NC組(1)及穩(wěn)轉(zhuǎn)miR-301a組(2)CFPAC-1細(xì)胞相關(guān)蛋白的表達(dá)

圖5　轉(zhuǎn)染miR-301a-NC組0、12、18 h(5A、5B、5C)及穩(wěn)轉(zhuǎn)miR-301a組0、12、18 h(5D、5E、5F)的細(xì)胞遷移狀況(劃痕實驗，×100)

五、穩(wěn)轉(zhuǎn)細(xì)胞侵襲能力的變化

穩(wěn)轉(zhuǎn)miR-301a組、轉(zhuǎn)染miR-301a-NC組的穿膜細(xì)胞數(shù)分別為(95±11)、(25±6)/100倍視野(圖6)，穩(wěn)轉(zhuǎn)細(xì)胞為轉(zhuǎn)染miR-301a-NC細(xì)胞的3.8倍，差異有統(tǒng)計學(xué)意義(t=9.68，P<0.05)。

圖6　轉(zhuǎn)染miR-301a-NC組(6A)、穩(wěn)轉(zhuǎn)miR-301a組(6B)的穿膜細(xì)胞(×100)

討論

胰腺癌是惡性程度極高的消化系統(tǒng)腫瘤之一，其發(fā)病率近年來呈上升趨勢。胰腺癌手術(shù)風(fēng)險高、難度大，術(shù)后遠(yuǎn)期生存率偏低[14-15]。近年來多項研究證實miRNA在腫瘤發(fā)生、癌細(xì)胞浸潤和遠(yuǎn)處轉(zhuǎn)移中具有重要作用。

有研究報道m(xù)iR-301高表達(dá)是乳腺癌一項不良預(yù)后指標(biāo)[16]，miR-301可靶向調(diào)控多個腫瘤相關(guān)基因(FOXF2、PTEN、BCL-2等)從而影響乳腺癌細(xì)胞的增殖、侵襲、轉(zhuǎn)移和血管生成等腫瘤生物學(xué)行為。其他研究亦表明FOXF2在乳腺癌細(xì)胞EMT的過程中發(fā)揮重要作用，F(xiàn)OXF2缺失可促進(jìn)TWST1表達(dá)進(jìn)而促進(jìn)乳腺癌細(xì)胞EMT和轉(zhuǎn)移[17]。

另有研究發(fā)現(xiàn)，在胃癌中miR-301a高表達(dá)與腫瘤大小、浸潤深度、淋巴結(jié)轉(zhuǎn)移和TNM分期密切相關(guān)[12]；在結(jié)直腸癌中miR-301a 表達(dá)升高可促進(jìn)腫瘤細(xì)胞增殖、侵襲和轉(zhuǎn)移[10]。miR-301a可能作為癌基因在腫瘤的發(fā)生發(fā)展過程中發(fā)揮重要功能。

本研究在低氧環(huán)境下培養(yǎng)胰腺癌細(xì)胞CFPAC-1，構(gòu)建低氧誘導(dǎo)胰腺癌細(xì)胞EMT模型。低氧培養(yǎng)2 d和4 d后CFPAC-1細(xì)胞發(fā)生明顯的形態(tài)學(xué)改變，由外形較規(guī)則、偽足較少、呈圓形或橢圓形、細(xì)胞間連接緊密的上皮型細(xì)胞向外形不規(guī)則、多呈棱形、偽足較多、細(xì)胞連接松散的間質(zhì)型細(xì)胞轉(zhuǎn)變，上皮型標(biāo)志物E-cadherin、ZO-1、β-catenin表達(dá)下調(diào)，而間質(zhì)性標(biāo)志物Vimentin、N-cadherin、Fibronectin表達(dá)上調(diào)，同時低氧培養(yǎng)的CFPAC-1細(xì)胞miR-301a表達(dá)量較常氧培養(yǎng)的CFPAC-1細(xì)胞的表達(dá)量明顯升高，提示miR-301a可能在低氧誘導(dǎo)的胰腺癌細(xì)胞 EMT的過程中發(fā)揮重要作用。本研究進(jìn)一步構(gòu)建miR-301a穩(wěn)定表達(dá)細(xì)胞系。穩(wěn)轉(zhuǎn)miR-301a的CFPAC-1細(xì)胞由上皮細(xì)胞形態(tài)向間質(zhì)細(xì)胞形態(tài)改變，E-cadherin、ZO-1、β-catenin表達(dá)下調(diào)， Vimentin、N-cadherin、Fibronectin表達(dá)上調(diào)，且體外遷移侵襲能力明顯提高，表明上調(diào)miR-301a可明顯促進(jìn)胰腺癌細(xì)胞EMT過程。

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(本文編輯：屠振興)

The effect of hypoxic culture on miR-301a expression in pancreatic cancer CFPAC-1 cells

ZhuLin,ZhangKundong,XiaXiang,HuangChen,QiuZhengjun.

DepartmentofGeneralSurgery,FirstPeople′sHospitalofShanghai,ShanghaiJiaotongUniversity,Shanghai200080,China

【Abstract】ObjectiveTo study the regulation on miR-301a expression in pancreatic cancer CFPAC-1 cells by low oxygen condition and explore the potential molecular mechanism of miR-301a. MethodsCFPAC-1 cells were cultured in 1% O2 and the morphology of CFPAC-1 cells was observed by phase contrast microscopy. The expression of HIF-1a, miR-301a, its′ target gene FOXF2 and EMT markers Vinentin, N-Cadherin and Fibronectin were detected by Western blot. The lentivirus vector expressing miR-301a was constructed and stably transfected CFPAC-1 cells were established using transfected with miR-301a-NC as control. After the transfection, the cell morphology was observed, and FOXF2 and the EMT markers expression were assessed.The migration and invasion of CFPAC-1 cells was determined by Transwell migration assay and wound healing test. ResultsThe CFPAC-1 cells after being cultured in hypoxic environment had an irregular shape with more fusiform and pseudopodia, loose intercellular connections, and were transformed into mesenchymal cell morphology. By setting the expression of target genes in cells cultured in normoxia as 1, the relative expression of miR-301a was 2.82±0.01 after being cultured in hypoxia for 4 d, and HIF-1a level was 2.17±0.36, and Vimentin, N-cadherin and Fibronectin level was 5.48±0.16, 1.16±0.03 and 1.30±0.03, respectively, which were significantly upregulated. And FOXF2, E-cadherin, ZO-1,β-catenin expression was 0.45±0.049, 0.61±0.049, 0.45±0.021, 0.37±0.02 respectively, which were significantly downregulated (all P<0.01). By setting the protein expression of target genes in cells transfected with miR-301a-NC as 1, the expression of miR-301a, Fibronectin, Vimentin and N-cadherin in cells transfected with miR-301a were 7.09±0.42、1.56±0.10、1.24±0.05、1.37±0.01, respectively，which were significantly upregulated. And the expression of FOXF2、ZO-1、E-cadherin、β-catenin was 0.33±0.03, 0.71±0.02, 0.67±0.06 and 0.47±0.03, respectively, which were significantly downregulated (all P<0.01). Results of scratch test showed that the healing rates of scratch area in miR-301a group after 18h was(94.82±2.18)%, which was significantly larger than that[(54.68±4.31)%] in miR-301a-NC group (P<0.01). The number of migration cells in miR-301a group and miR-301a-NC group was 95±11 and 25±6 at 100×magnification, respectively. Compared with control group, the cells that migrated through membrane were obviously increased in the group transfected with miR-301a (P<0.01). ConclusionsEMT and upregulation of miR-301a were observed in CFPAC-1 cells after being cultured in low oxygen. miR-301a may promote EMT by inhibiting FOXF2 expression in pancreatic cancer.

【Key words】Pancreatic neoplasms;Anoxia; Epithelial mesenchymal transformation;MicroRNAs

DOI:10.3760/cma.j.issn.1674-1935.2016.03.005

通信作者：裘正軍，Email: qiuzjdoctor@sina.com

基金項目：國家自然科學(xué)基金(81372640)

Corresponding author:Qiu Zhengjun, Email: qiuzjdoctor@sina.com

(收稿日期：2015-11-04)