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細胞miR-301a表達的影響

朱麟張坤東夏翔黃陳裘正軍

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

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

【關鍵詞】胰腺腫瘤；缺氧；上皮間質轉化；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)境是誘導胰腺癌高侵襲性的重要因素[1]。上皮間質轉化(EMT)是上皮細胞來源的腫瘤細胞侵襲轉移的一個重要步驟。Cheng等[2]的研究證實，缺氧可誘導HIF-1α的表達從而導致胰腺癌細胞發(fā)生EMT，促進胰腺癌的侵襲轉移。近年來很多研究證實，miRNA參與腫瘤細胞EMT及轉移過程[3-7]。miR-301a在乳腺癌[8]、肝癌[9]、結直腸癌[10]、胃癌[11-12]以及胰腺癌[13]中表達升高，在腫瘤增殖、凋亡、血管生成和侵襲轉移中發(fā)揮重要作用，然而miR-301a與胰腺癌進展的相關性尚不明確，其在胰腺癌EMT中的作用尚未見報道。本研究初步探討miR-301a在低氧誘導胰腺癌細胞EMT中的作用及其相關機制。

材料與方法

一、細胞培養(yǎng)

人胰腺癌細胞系CFPAC-1購自美國ATCC細胞庫。細胞復蘇后應用含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胞形態(tài)。

二、實時PCR

三、蛋白質印跡法

用SDS裂解液提取細胞蛋白，煮沸變性后常規(guī)行蛋白質印跡法檢測細胞低氧誘導因子HIF-1α、miR-301a及其靶基因FOXF2，上皮型標志物E-cadherin、ZO-1、β-catenin，上皮間質轉化標志物Vimentin、Fibronection、N-cadherin蛋白表達，以β-actin為內(nèi)參。抗E-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量。

四、miR-301a穩(wěn)轉細胞株建立

應用Primer 5軟件設計miR-301a擴增引物，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。

采用脂質體2000(Life Technologies公司)將LV-miR-301a-mCherry和包裝質粒pMDG、pCMV△8.9共轉染293T細胞，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細胞48 h，應用嘌呤霉素篩選miR-301a穩(wěn)定轉染的細胞。

五、劃痕實驗

收集穩(wěn)轉miR-301a的CFPAC-1細胞接種于6孔板，每孔約5×105個細胞，培養(yǎng)至細胞鋪滿后用槍頭垂直劃線，PBS洗滌3次去除劃下的細胞，加入培養(yǎng)液分別繼續(xù)培養(yǎng)12、18 h，以轉染miR-301a-NC的細胞作為對照組。觀察細胞遷移覆蓋劃痕區(qū)的情況。實驗重復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項目，應當依法進行環(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miR-301a的CFPAC-1細胞，用無血清培養(yǎng)基重懸，上室加入200 μl細胞懸液(5×104個細胞)，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胞，甲醇固定15 min，結晶紫染色3 h，100倍顯微鏡下計算4個視野的穿膜細胞數(shù)，取均值。以轉染miR-301a-NC的細胞作為對照組。

七、統(tǒng)計學處理

結果

一、低氧培養(yǎng)下CFPAC-1細胞的形態(tài)改變

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

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

二、低氧培養(yǎng)后CFPAC-1細胞相關蛋白表達的變化(圖2)

低氧培養(yǎng)后2、4 d，細胞miR-301a表達為常規(guī)培養(yǎng)細胞的(2.71±0.04)、(2.82±0.01)倍，差異有統(tǒng)計學意義(t值分別為14.74、28.72，P值均<0.01)。以常氧培養(yǎng)細胞的蛋白表達量為1，低氧培養(yǎng)后2、4 d時細胞HIF-1α相對表達量為4.11±0.60、2.17±0.36，差異有統(tǒng)計學意義(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)計學意義(P值均<0.01)。

三、穩(wěn)轉miR-301a的CFPAC-1細胞

常氧培養(yǎng)下轉染miR-301a-NC細胞呈上皮細胞形態(tài)(圖3A)，轉染miR-301a細胞呈間質細胞形態(tài)改變(圖3B)。

圖2　常氧(1)及低氧組2、4 d(2、3)CFPAC-1細胞相關蛋白的表達

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

轉染miR-301a細胞的miR-301a表達量為轉染miR-301a-NC細胞的(7.09±0.42)倍(t=14.47，P<0.01)。以轉染miR-301a-NC細胞的蛋白表達量為1，轉染miR-301a細胞的FOXF2、ZO-1、E-cadherin、β-catenin相對表達量分別為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)計學意義(P值均<0.01，圖4)。

四、穩(wěn)轉細胞遷移能力的變化

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

圖4　轉染miR-301a-NC組(1)及穩(wěn)轉miR-301a組(2)CFPAC-1細胞相關蛋白的表達

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

五、穩(wěn)轉細胞侵襲能力的變化

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

圖6　轉染miR-301a-NC組(6A)、穩(wěn)轉miR-301a組(6B)的穿膜細胞(×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。胰腺癌手術風險高、難度大，術后遠期生存率偏低[14-15]。近年來多項研究證實miRNA在腫瘤發(fā)生、癌細胞浸潤和遠處轉移中具有重要作用。

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

另有研究發(fā)現(xiàn)，在胃癌中miR-301a高表達與腫瘤大小、浸潤深度、淋巴結轉移和TNM分期密切相關[12]；在結直腸癌中miR-301a 表達升高可促進腫瘤細胞增殖、侵襲和轉移[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)胰腺癌細胞CFPAC-1，構建低氧誘導胰腺癌細胞EMT模型。低氧培養(yǎng)2 d和4 d后CFPAC-1細胞發(fā)生明顯的形態(tài)學改變，由外形較規(guī)則、偽足較少、呈圓形或橢圓形、細胞間連接緊密的上皮型細胞向外形不規(guī)則、多呈棱形、偽足較多、細胞連接松散的間質型細胞轉變，上皮型標志物E-cadherin、ZO-1、β-catenin表達下調(diào)，而間質性標志物Vimentin、N-cadherin、Fibronectin表達上調(diào)，同時低氧培養(yǎng)的CFPAC-1細胞miR-301a表達量較常氧培養(yǎng)的CFPAC-1細胞的表達量明顯升高，提示miR-301a可能在低氧誘導的胰腺癌細胞 EMT的過程中發(fā)揮重要作用。本研究進一步構建miR-301a穩(wěn)定表達細胞系。穩(wěn)轉miR-301a的CFPAC-1細胞由上皮細胞形態(tài)向間質細胞形態(tài)改變，E-cadherin、ZO-1、β-catenin表達下調(diào)， Vimentin、N-cadherin、Fibronectin表達上調(diào)，且體外遷移侵襲能力明顯提高，表明上調(diào)miR-301a可明顯促進胰腺癌細胞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：國家自然科學基金(81372640)

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

(收稿日期：2015-11-04)