陳 潔,嚴(yán)曉娣,張東升,錢紅運(yùn),陳瑞嬌,周方正
干預(yù)FoxM1表達(dá)對(duì)食管癌細(xì)胞增殖、周期及凋亡的影響
陳 潔1,嚴(yán)曉娣2,張東升1,錢紅運(yùn)3,陳瑞嬌3,周方正1
目的探討干預(yù)FoxM1表達(dá)對(duì)食管癌細(xì)胞增殖、細(xì)胞周期及凋亡的影響。方法選擇FoxM1高表達(dá)食管癌細(xì)胞株,構(gòu)建FoxM1-shRNA干擾質(zhì)粒轉(zhuǎn)染食管癌細(xì)胞,觀察干預(yù)FoxM1表達(dá)對(duì)食管癌細(xì)胞增殖、細(xì)胞周期及凋亡的影響。結(jié)果成功轉(zhuǎn)染后細(xì)胞中FoxM1蛋白水平表達(dá)明顯降低(t=13.17,P<0.01),細(xì)胞增殖明顯受抑并呈時(shí)間依賴性,72 h抑制效果最顯著(68.0%±6.4%,P<0.05);干預(yù)組細(xì)胞增殖周期發(fā)生G1期阻滯(59.14%±1.69%vs40.51%±1.45%,t=14.23,P<0.01)、細(xì)胞凋亡增加(2.48%±0.49%vs35.37%±0.56%,t=76.56,P<0.01)。結(jié)論干預(yù)FoxM1表達(dá)使細(xì)胞周期發(fā)生G1期阻滯并能促進(jìn)細(xì)胞凋亡,從而抑制TE1細(xì)胞增殖,F(xiàn)oxM1可能是食管癌基因治療潛在的有效靶點(diǎn)。
食管癌;叉頭框轉(zhuǎn)錄因子M1;基因沉默;細(xì)胞增殖;細(xì)胞凋亡
食管癌是最常見的惡性腫瘤之一,列腫瘤相關(guān)死亡因素第三位,全球每年新發(fā)病例約48萬,死亡病例約40萬,且發(fā)病率呈逐年上升的趨勢(shì)[1]。我國是全球食管癌高發(fā)地區(qū)之一,以食管鱗癌(esophageal squamous cell carcinoma,ESCC)占絕大多數(shù)[1, 2]。盡管采取積極手術(shù)、放化療等多途徑聯(lián)合治療,但絕大多數(shù)患者仍死于腫瘤局部復(fù)發(fā)及遠(yuǎn)處轉(zhuǎn)移[3]。
新近研究發(fā)現(xiàn)叉頭框轉(zhuǎn)錄因子M1 ( forkhead box protein M1, FoxM1)作為一種增生相關(guān)的轉(zhuǎn)錄因子,在食管癌細(xì)胞中表達(dá)具有一定特異性及敏感性,被認(rèn)為是食管癌特異性治療的潛在靶點(diǎn)[4]。本文采用RNA干擾(RNA interference, RNAi)技術(shù),以特異性shRNA下調(diào)FoxM1表達(dá),觀察其對(duì)食管癌細(xì)胞增殖、周期等生物學(xué)活性的影響,探討其靶向治療價(jià)值,為食管癌靶向治療提供理論依據(jù)。
1.1 細(xì)胞株來源與細(xì)胞培養(yǎng) 正常食管上皮細(xì)胞株(HEEC)及食管癌細(xì)胞株(Eca109、TE1、TE7、TE2)均購自中國科學(xué)院上海生命科學(xué)研究院生物化學(xué)與細(xì)胞生物學(xué)研究所。用含10%胎牛血清的DMEM培養(yǎng)基培養(yǎng)液(美國Invitrogen公司),在孵箱(37 ℃、5%CO2)中貼壁培養(yǎng),細(xì)胞基本鋪滿瓶底時(shí)傳代。
1.2 細(xì)胞轉(zhuǎn)染及篩選 依據(jù)GenJetTM DNA In Vitro Transfection Reagent (美國SignaGen公司)試劑說明書進(jìn)行轉(zhuǎn)染。細(xì)胞密度達(dá)到80%左右時(shí),將細(xì)胞分為空白組(Untreated)、陰性組(shRNA-Neg)及干預(yù)組(shRNA),以GenJetTM轉(zhuǎn)染試劑分別將FoxM1-shRNA或neg-shRNA轉(zhuǎn)染至食管癌細(xì)胞。轉(zhuǎn)染48 h后在倒置熒光顯微鏡下計(jì)算轉(zhuǎn)染效率并收集細(xì)胞備用。
1.3 蛋白質(zhì)印跡法 收集足夠數(shù)量細(xì)胞依據(jù)試劑說明書進(jìn)行蛋白抽提。每泳道加50 μg蛋白,在10%SDS 聚丙烯酰胺凝膠電泳,轉(zhuǎn)膜,封閉,加入兔抗人FoxM1(1∶500)或β-actin抗體(1∶1000),β-actin為內(nèi)參,4 ℃過夜,辣根過氧化物酶標(biāo)記的羊抗兔IgG二抗(1∶1000) 37 ℃孵育1 h,洗滌后ECL顯色并拍照。
1.4 CCK-8法檢測(cè)細(xì)胞增殖 細(xì)胞轉(zhuǎn)染48 h后收集細(xì)胞,分別接種于96孔板(n=5),每組分調(diào)零、空白、陰性及干預(yù)孔,每組含復(fù)孔5個(gè),分別在規(guī)定的時(shí)間點(diǎn)添加CCK-8試劑(日本同仁化學(xué)研究所),培養(yǎng)4 h,脫色搖床1 min,酶標(biāo)儀檢測(cè)A450值。
1.5 細(xì)胞周期分析 轉(zhuǎn)染48 h后,換新鮮完全培養(yǎng)液繼續(xù)培養(yǎng)24 h后收集細(xì)胞,在-20 ℃ 70%預(yù)冷乙醇固定過夜,用預(yù)冷PBS洗滌沉淀后加0.5 ml碘化丙啶染色,避光溫浴30 min(37 ℃),再冰浴避光存放;流式細(xì)胞儀檢測(cè)紅色熒光(488 nm波長),利用Multicycle軟件(Beckman Coulter公司)收集、存儲(chǔ)和分析數(shù)據(jù)。
1.6 Annexin V-PE/7-ADD雙染檢測(cè)細(xì)胞凋亡 轉(zhuǎn)染48 h后換新鮮完全培養(yǎng)液繼續(xù)培養(yǎng)24 h后收集細(xì)胞,加入500 μl緩沖液懸浮細(xì)胞,再加1 μl Annexin V-PE混勻,室溫避光反應(yīng)15 min;最后加5 μl 7-ADD,輕輕混勻,室溫避光反應(yīng)15 min后用流式細(xì)胞儀檢測(cè)分析結(jié)果。
2.1 FoxM1蛋白在不同食管癌細(xì)胞株中表達(dá)情況 FoxM1/β-actin灰度強(qiáng)度比值分別為0.15±0.04、0.61±0.11、0、0.03±0.09及0.48±0.05;FoxM1蛋白在正常食管上皮細(xì)胞株中幾乎不表達(dá),在食管癌細(xì)胞株(Eca109、TE1、TE2、TE7)中呈不同程度表達(dá)(圖1),其中TE1細(xì)胞株中表達(dá)水平最高,選擇TE1細(xì)胞株用于后續(xù)研究。
圖1 Western blotting分析不同食管癌細(xì)胞中FoxM1蛋白表達(dá)(n=3)
2.2 FoxM1-shRNA轉(zhuǎn)染食管癌細(xì)胞 倒置熒光顯微鏡下成功轉(zhuǎn)染的細(xì)胞帶綠色熒光(圖2),轉(zhuǎn)染效率為93%(n=5)。
圖2 FoxM1-shRNA轉(zhuǎn)染食管癌細(xì)胞后普通光鏡與熒光顯微鏡圖(×200)
2.3 FoxM1-shRNA下調(diào)食管癌細(xì)胞FoxM1表達(dá)水平 空白組(Untreated)、陰性組(shRNA-Neg)及干預(yù)組(shRNA)FoxM1/β-actin灰度強(qiáng)度比值分別為0.39±0.07、0.41±0.02和0.07±0.04(t=13.17,P<0.01),陰性組與空白組間未見統(tǒng)計(jì)學(xué)差異(t=0.48,P>0.05),見圖3。
圖3 shRNA下調(diào)食管癌細(xì)胞中FoxM1蛋白表達(dá)水平(n=3)
2.4 FoxM1-shRNA抑制食管癌細(xì)胞增殖 FoxM1-shRNA組24 h、48 h及72 h生長抑制率分別為(49.0±6.3)%、(56.0±4.5)%及(68.0±6.4)%,抑制作用呈時(shí)間依賴性,72 h作用最顯著。
2.5 FoxM1-shRNA影響食管癌細(xì)胞增殖周期 干預(yù)組G1期細(xì)胞明顯增多(t=14.23,P<0.01),S期細(xì)胞減少(t=14.08,P<0.01),G2期改變無統(tǒng)計(jì)學(xué)差異,陰性組與空白組間未見明顯差異。
表1 流式細(xì)胞術(shù)分析細(xì)胞周期含量改變 (n=3;;%)
注:與空白組比較,①P<0.01;與陰性組比較,②P<0.01
2.6 FoxM1-shRNA促進(jìn)食管癌細(xì)胞凋亡 空白組、陰性組及干預(yù)組細(xì)胞凋亡率分別為(2.48±0.49)%、(2.56±0.71)%及(35.37±0.56)%,干預(yù)組細(xì)胞凋亡率顯著增高(t=76.56,P<0.01),陰性組與空白組間未見統(tǒng)計(jì)學(xué)差異。
手術(shù)及放、化療是食管癌的主要治療手段,但是其高復(fù)發(fā)率及轉(zhuǎn)移率,導(dǎo)致ESCC預(yù)后極差。尋找更加有效的早期診斷及治療方法是當(dāng)前亟待解決的問題。
ESCC發(fā)生發(fā)展是一個(gè)復(fù)雜的多階段過程,但其具體分子機(jī)制尚不完全清楚。FoxM1是增生相關(guān)的轉(zhuǎn)錄因子,通過對(duì)多種基因轉(zhuǎn)錄調(diào)控實(shí)現(xiàn)G1/S轉(zhuǎn)換及G2/M轉(zhuǎn)換,促進(jìn)有絲分裂進(jìn)行,同時(shí)涉及DNA損傷修復(fù)、染色體組裝、蛋白合成等生物學(xué)過程,在生命現(xiàn)象中發(fā)揮重要作用[3, 5]。FoxM1在肺癌、乳腺癌、結(jié)腸癌、卵巢癌等多種惡性腫瘤中異常過度表達(dá)并與腫瘤發(fā)生及轉(zhuǎn)移密切相關(guān)[6-10],提示FoxM1可能在腫瘤增殖過程中扮演癌基因的角色。已有研究發(fā)現(xiàn)FoxM1在食管癌細(xì)胞中表達(dá)具有一定特異性及敏感性,被認(rèn)為是食管癌特異治療的潛在靶點(diǎn),本文采用RNAi技術(shù)[11, 12]構(gòu)建FoxM1-shRNA質(zhì)粒,特異性抑制食管癌細(xì)胞FoxM1表達(dá),研究對(duì)細(xì)胞增殖、細(xì)胞周期及凋亡等生物學(xué)行為的影響,初步探討其應(yīng)用于靶向治療的可能。
FoxM1蛋白在4種食管癌細(xì)胞株(Eca109、TE1、TE2及TE7)中呈差異表達(dá),其中TE1細(xì)胞株中表達(dá)水平最高,選擇TE1細(xì)胞株用于后續(xù)研究,與課題組前期研究FoxM1在食管鱗癌組織中呈高表達(dá)而在癌周正常組織不表達(dá)結(jié)果相一致。構(gòu)建特異性干擾序列FoxM1-shRNA成功轉(zhuǎn)染至TE1細(xì)胞,干預(yù)組FoxM1蛋白水平降低,提示該質(zhì)粒在體外能夠干預(yù)FoxM1表達(dá),符合設(shè)計(jì)要求,用于后續(xù)實(shí)驗(yàn)。
觀察干預(yù)FoxM1表達(dá)對(duì)TE1細(xì)胞增殖、周期及凋亡等生物學(xué)行為的影響。干預(yù)組細(xì)胞增殖受到抑制,呈時(shí)間依賴性,提示干預(yù)FoxM1能夠抑制TE1細(xì)胞增殖,該作用呈時(shí)間依賴性,與文獻(xiàn)[4]相關(guān)報(bào)道一致。為探討干預(yù)FoxM1所產(chǎn)生的生長抑制效應(yīng)是否涉及細(xì)胞凋亡及細(xì)胞周期進(jìn)程的改變,利用流式細(xì)胞儀檢測(cè)干預(yù)后細(xì)胞DNA含量分布,干預(yù)組G1期細(xì)胞增多,S期細(xì)胞減少,G2期改變不明顯,提示細(xì)胞增殖周期發(fā)生G1期阻滯;利用流式細(xì)胞儀檢測(cè)干預(yù)后細(xì)胞分布,干預(yù)組細(xì)胞凋亡率顯著增高。
關(guān)于FoxM1在其他惡性腫瘤中的研究發(fā)現(xiàn)過表達(dá)FoxM1分子的腫瘤細(xì)胞,在敲除FoxM1后,腫瘤細(xì)胞增殖、抗凋亡及侵襲能力顯著受抑[13-16]。研究發(fā)現(xiàn)呼吸道上皮細(xì)胞中FoxM1的表達(dá)是肺癌形成及TOPO-2α表達(dá)的關(guān)鍵因素,在MCA/BHT誘導(dǎo)腫瘤形成之前,選擇性敲除FoxM1基因的轉(zhuǎn)基因小鼠FoxM1-/-的肺癌數(shù)量及體積均顯著下降,這可能與TOPO-2α(調(diào)節(jié)腫瘤細(xì)胞增殖的關(guān)鍵因子)基因表達(dá)下調(diào)有關(guān),F(xiàn)oxM1識(shí)別其啟動(dòng)子序列,結(jié)合并誘導(dǎo)其表達(dá),間接促進(jìn)腫瘤細(xì)胞的增殖;后續(xù)研究證實(shí)肺部腫瘤形成后FoxM1缺失可延緩腫瘤的進(jìn)展,進(jìn)一步敘述了FoxM1促進(jìn)腫瘤發(fā)生發(fā)展的機(jī)制[17]。應(yīng)用RNA干擾技術(shù)使乳腺癌的MDA-MB-231細(xì)胞系的癌細(xì)胞內(nèi)轉(zhuǎn)入PEI相關(guān)的物質(zhì)后可導(dǎo)致癌細(xì)胞內(nèi)FOXM1表達(dá)水平下調(diào),同時(shí)癌細(xì)胞內(nèi)蛋白表達(dá)水平下降,并且Cdc25B的mRNA的水平亦下降。本研究結(jié)果提示:在敲除FoxM1后,腫瘤細(xì)胞的增殖、抗凋亡及侵襲能力顯著受抑,表明調(diào)控FoxM1的表達(dá)水平,或許能控制腫瘤的生長。
綜上所述,干預(yù)FoxM1表達(dá)使細(xì)胞周期發(fā)生G1期阻滯并促進(jìn)細(xì)胞凋亡,從而抑制TE1細(xì)胞增殖,F(xiàn)oxM1可能是食管癌基因治療潛在的有效靶點(diǎn)。
[1] Lam E W, Brosens J J, Gomes A R,etal. Forkhead box proteins: tuning forks fortranscriptional harmony [J]. Nat Rev Cancer,2013, 13(7): 482-495.
[2] Koo C Y, Muir K W, Lam E W. FOXM1: from cancer initiation to progression andtreatment [J]. Biochim Biophys Acta, 2012, 1819(1): 28-37.
[3] Wierstra I, Alves J. FoxM1, a typical proliferation associated transcription factor [J]. Biol Chem, 2007, 388(12): 1257-1274.
[4] 蓋 領(lǐng),茅國新,劉 軍,等.叉頭框轉(zhuǎn)錄因子M1蛋白在食管鱗癌細(xì)胞和組織中的表達(dá)及意義[J]. 中華腫瘤雜志, 2016,38(3):179-184.
[5] Wu X, Gu X, Han X,etal. A novel function for FoxM1 in interkinetic nuclear migration in the developing telencephalon and anxiety-related behavior [J]. J Neurosci, 2014, 34(4): 1510-1522.
[6] Huang C, Xie D, Cui J,etal. FOXM1c promotes pancreatic cancer epithelial to mesenchymal transition and metastasis via upregulation of expression of the urokinase plasminogen activator system [J]. Clin Cancer Res, 2014, 20(6): 1477- 1488.
[7] Xia L, Huang W, Tian D,etal. Overexpression of forkhead box C1 promotes tumor metastasis and indicates poor prognosis in hepatocellular carcinoma [J]. Hepatology, 2013, 57(2): 610-624.
[8] Kong F F, Qu Z Q, Yuan H H,etal. Overexpression of FOXM1 is associated with EMT and is a predictor of poor prognosis in non-small cell lung cancer [J]. Oncol Rep, 2014, 31(6): 2660-2668.
[9] Arora R, Yates C, Gary B D,etal. Panepoxydone targets NF-kB and FOXM1 to inhibit proliferation, induce apoptosis and reverse epithelial to mesenchymal transition in breast cancer [J]. PLoS One, 2014, 9(6): e98370.
[10] Wen N, Wang Y, Wen L,etal. Overexpression of FOXM1 predicts poor prognosis and promotes cancer cell proliferation, migration and invasion in epithelial ovarian cancer [J]. J Transl Med, 2014, 12(134): 1-13.
[11] Cheng A L, Kang Y K, Chen Z,etal. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial [J]. Lancet Oncol, 2009, 10(1): 25-34.
[12] Lee Y L, Ahn B C, Lee Y,etal. Targeting of hepatocellular carcinoma with glypican-3 targeting peptide ligand [J]. J Pept Sci, 2011, 17(11): 763-769.
[13] Li X R, Chu H J, Lv T,etal. miR-342-3p suppresses proliferation, migration and invasion by targeting FOXM1 in human cervical cancer [J]. FEBS Lett, 2014, 588(17): 3298-3307.
[14] Zhou J, Wang Y, Wang Y,etal. FOXM1 modulates cisplatin sensitivity by regulating EXO1 in ovarian cancer [J]. PLoS One, 2014, 9(5): e96989.
[15] Miao L, Xiong X, Lin Y,etal. Down-regulation of FoxM1 leads to the inhibition of the epithelial-mesenchymal transition in gastric cancer cells [J]. Cancer Genet, 2014, 207(3): 75-82.
[16] Chen T, Xiong J, Yang C,etal. Silencing of FOXM1 transcription factor expression by adenovirus-mediated RNA interference inhibits human hepatocellular carcinoma growth [J]. Cancer Gene Ther, 2014, 21(3): 133-138.
[17] Wang I C, Meliton L, Ren X,etal. Deletion of Forkhead Box M1 transcription factor from respiratory epithelial cells inhibits pulmonary tumorigenesis [J]. PLoS One, 2009,4(8):e6609.
Effectsofdown-regulationofFoxM1expressionsonproliferationandapoptosisofesophagealsquamouscellcarcinomacells
CHEN Jie1, YAN Xiaodi2, ZHANG Dongsheng1, QIAN Hongyun3, CHEN Ruijiao3, and ZHOU Fangzheng1.
1.Oncology Department of Suizhou Central Hospital, Affiliated Hospital of Hubei University of Medicine, Suizhou 441300, China;2.Tumor Radiotherapy Department of Affiliated Hospital of Nantong University, Nantong 22600, China; 3. Clinical Medicine Department of Hubei University of Medicine, Shiyan 442000, China
ObjectiveTo investigate the effects of down-regulation of forkhead box protein M1 (FoxM1) expressions by shRNA on proliferation and apoptosis of esophageal squamous cell carcinoma (ESCC) cells.MethodsPlasmid that highly expressed FoxM1-shRNA was successfully constructed and transfected with TE1 cell line. FoxM1 was analyzed by Western blot. Cell proliferation was tested by cell counting kit-8 while cell cycle and apoptosis were analyzed by flow cytometry and Annexin-V-PE/7-AAD staining kit.ResultsFoxM1 expressions in ESCC cells transfected with specific shRNA were significantly down-regulated at the protein level and cell proliferation was inhibited in a time-dependent manner. The inhibition rate was 68.0%±6.4% (P<0.05), with cell cycle arrested at G1phase arrest (59.14%±1.69%vs40.51%±1.45%,t=14.23,P<0.01)and the apoptosis rate of TE1 cells reached 35.37% (2.48%±0.49%vs35.37%±0.56%,t=76.56,P<0.01).ConclusionsInterventions with FoxM1 gene expressions can inhibit the proliferation and apoptosis of ESCC cells, suggesting that FoxM1 can be a novel therapeutic target for ESCC.
esophageal squamous cell carcinoma; forkhead box protein M1; gene silencing; cell proliferation; apoptosis
2016年湖北省教育廳科學(xué)研究計(jì)劃指導(dǎo)性項(xiàng)目(B2016120),2015年隨州市科研立項(xiàng)(2015SMR003),2016年隨州市科研立項(xiàng)(2016SZ32005)。
陳 潔,碩士,住院醫(yī)師。
1.441300, 湖北醫(yī)藥學(xué)院附屬隨州醫(yī)院 隨州市中心醫(yī)院腫瘤科;2.226001, 南通大學(xué)附屬醫(yī)院放療科;3. 422000 十堰,湖北醫(yī)藥學(xué)院臨床醫(yī)學(xué)系
周方正,E-mail: 918771802@qq.com
R735.1
(2017-06-02收稿 2017-07-16修回)
(責(zé)任編輯 武建虎)