叉頭框蛋白3過(guò)表達(dá)對(duì)胃癌細(xì)胞生物學(xué)行為的影響

馬桂芬， 潘獨(dú)伊， 何 健， 曾昭沖， 陳世耀*

1.復(fù)旦大學(xué)附屬中山醫(yī)院放療科， 上海 200032 2.復(fù)旦大學(xué)附屬中山醫(yī)院消化內(nèi)科， 上海 200032

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·論 著·

叉頭框蛋白3過(guò)表達(dá)對(duì)胃癌細(xì)胞生物學(xué)行為的影響

馬桂芬1， 潘獨(dú)伊2， 何 健1， 曾昭沖1， 陳世耀2*

1.復(fù)旦大學(xué)附屬中山醫(yī)院放療科， 上海 200032 2.復(fù)旦大學(xué)附屬中山醫(yī)院消化內(nèi)科， 上海 200032

目的： 探討叉頭框蛋白3(Forkhead box protein 3，F(xiàn)oxP3)過(guò)表達(dá)對(duì)胃癌細(xì)胞生物學(xué)行為的影響。方法： 用shRNA過(guò)表達(dá)質(zhì)粒轉(zhuǎn)染胃癌細(xì)胞，經(jīng)藥物篩選和單克隆挑選，建立穩(wěn)定轉(zhuǎn)染的細(xì)胞系。用四甲基偶氮唑鹽(MTT)法檢測(cè)轉(zhuǎn)染FoxP3基因后胃癌細(xì)胞系生長(zhǎng)增殖能力的改變；流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期；MTT法測(cè)定細(xì)胞轉(zhuǎn)染FoxP3基因后對(duì)化療藥物敏感性的影響。結(jié)果： 成功建立穩(wěn)定轉(zhuǎn)染FoxP3基因的胃癌細(xì)胞。與空載體轉(zhuǎn)染的對(duì)照組相比，上調(diào)FoxP3基因的胃癌細(xì)胞生長(zhǎng)增殖能力減慢,遷移能力減弱[穿膜細(xì)胞數(shù)：(203±42)個(gè)/HPvs(891±100)個(gè)/HP,P<0.05],對(duì)化療藥物的敏感性增加(P<0.05)。結(jié)論： 過(guò)表達(dá)FoxP3基因可抑制胃癌細(xì)胞的增殖、遷移，提高化療敏感性。

FoxP3； 胃癌； 基因功能； 生物學(xué)行為

2012年全球惡性腫瘤統(tǒng)計(jì)中，胃癌發(fā)病率排名第5位，在亞洲國(guó)家發(fā)病率更高達(dá)24.1 /10萬(wàn)，死亡率居第3位[1]。我國(guó)現(xiàn)有胃癌患者約300萬(wàn)，嚴(yán)重威脅人們的健康。目前對(duì)胃癌主要采用以手術(shù)為主，放療、化療為輔的綜合治療，雖然療效較前有很大的改善，但中位生存期僅14.6個(gè)月，3年生存率僅27%[2]。分子靶向治療能從根本上改變癌癥的發(fā)生發(fā)展，糾正患者機(jī)體的促癌狀態(tài)，有望成為有效的胃癌治療新途徑。

人類叉頭框蛋白3(Forkhead box protein 3, FoxP3)基因突變會(huì)導(dǎo)致致死性疾病，如免疫功能紊亂(immune dysregulation)、多腺體病(polyendocrinopathy)、腸病(enteropathy)和X染色體連鎖綜合征(X-linked syndrome，IPEX綜合征)[3]。近年來(lái)發(fā)現(xiàn)，F(xiàn)oxP3表達(dá)于腫瘤細(xì)胞中，可能是一種新的腫瘤調(diào)控因子。在腫瘤微環(huán)境中，腫瘤細(xì)胞可能通過(guò)表達(dá)FoxP3 抑制免疫活性,以此來(lái)模擬Treg細(xì)胞的功能[4]。FoxP3陽(yáng)性的胰腺癌細(xì)胞系可抑制CD4+T細(xì)胞的擴(kuò)增，可能與腫瘤細(xì)胞免疫逃逸有關(guān)[5]。目前大部分腫瘤中FoxP3表達(dá)增加，如小細(xì)胞肺癌[6]、肝癌[7]和胃癌[8]等。然而FoxP3基因?qū)ξ赴┘?xì)胞生物學(xué)行為的影響目前尚不清楚。

1 材料與方法

1.1 重組質(zhì)粒DNA轉(zhuǎn)染胃癌細(xì)胞 AGS胃癌細(xì)胞株購(gòu)于中國(guó)科學(xué)院上海生命科學(xué)院細(xì)胞庫(kù)。用含10% 胎牛血清(FBS)及100 U/mL青霉素和0.1 mg/mL鏈霉素的DMEM完全培養(yǎng)基(美國(guó)Gibco公司)于37℃、5% CO2的培養(yǎng)箱中培養(yǎng)。取對(duì)數(shù)生長(zhǎng)期的細(xì)胞，調(diào)整密度至2×105個(gè)/mL，接種至12孔培養(yǎng)板，培養(yǎng)至細(xì)胞融合度達(dá)70%～75%。將pEGFP/FoxP3和pEGFP/vector shRNA質(zhì)粒(由上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司構(gòu)建)分別與Lipofectamine 2000 (美國(guó)Invitrogen公司) 混合后，用Opti-MEMI無(wú)血清培養(yǎng)基(美國(guó)Gibco公司)稀釋至50 μL。將混合液加入培養(yǎng)板進(jìn)行轉(zhuǎn)染,6 h后換液，轉(zhuǎn)染48 h后，收集細(xì)胞，按1∶10接種于培養(yǎng)皿中,使用含G418的選擇培養(yǎng)基進(jìn)行抗性篩選，每3 d更換培養(yǎng)基。3周后挑取細(xì)胞克隆，擴(kuò)大培養(yǎng)。轉(zhuǎn)染成功的陽(yáng)性細(xì)胞克隆在熒光顯微鏡下可觀察到綠色熒光，挑選出增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)表達(dá)最強(qiáng)的單個(gè)細(xì)胞克隆。進(jìn)一步采用Western 印跡法選取FoxP3蛋白表達(dá)最強(qiáng)的克隆，擴(kuò)大培養(yǎng)后用于后續(xù)實(shí)驗(yàn)。

1.2 流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期 細(xì)胞周期試劑盒購(gòu)于南京凱基生物科技發(fā)展有限公司。取對(duì)數(shù)生長(zhǎng)期細(xì)胞，倒去培養(yǎng)液，胰酶適度消化，用培養(yǎng)液吹打，250 ×g離心 15 min,去上清；PBS洗2次，加0.5 mL 磷酸緩沖鹽溶液(PBS)混勻，務(wù)必吹散；用5 mL注射器將細(xì)胞吸起，用力打入5 mL 70%乙醇中，固定過(guò)夜；第2天收集固定的細(xì)胞，PBS洗2次；加RNase A約3 μL至終濃度約為50 μg/mL，37℃水浴消化30 min；加碘化丙啶(PI)約50 μL至終濃度約為65 μg/mL，在冰浴中避光染色30 min；用流式細(xì)胞儀(美國(guó)BD公司)檢測(cè)細(xì)胞周期。

1.3 四甲基偶氮唑鹽(MTT)法檢測(cè)細(xì)胞增殖能力 MTT檢測(cè)試劑盒購(gòu)于南京凱基生物科技發(fā)展有限公司。分別取對(duì)數(shù)生長(zhǎng)期的pEGFP/FoxP3及pEGFP/vector轉(zhuǎn)染后的AGS細(xì)胞，調(diào)整細(xì)胞密度為104個(gè)/mL，接種至96孔板，每孔200 μL，每組設(shè)3～5個(gè)復(fù)孔；分別培養(yǎng)24、48、72、96 h后,每孔加入5 mg/mL濃度的MTT試劑 20 μL，繼續(xù)培養(yǎng)4 h,棄培養(yǎng)液，加入150 μL DMSO，振蕩后用酶標(biāo)儀檢測(cè)550 nm處光密度(D)值，以24 h為基準(zhǔn)，計(jì)算以后每天的增長(zhǎng)倍數(shù)。

1.4 細(xì)胞遷移實(shí)驗(yàn) 分別取對(duì)數(shù)生長(zhǎng)期的AGS/FoxP3和AGS/vector細(xì)胞，調(diào)整其密度至5×105個(gè)/mL，接種至Transwell小室內(nèi)(美國(guó)Corning公司)，每孔200 μL；在下室加入0.5 mL含10%FBS的RPMI 1640完全培養(yǎng)基，放入培養(yǎng)箱培養(yǎng)；24 h后取出Transwell小室，用棉簽拭去上室內(nèi)細(xì)胞，置于預(yù)冷的無(wú)水乙醇中固定10 min；PBS洗滌3次后將Transwell小室浸入吉姆薩染色液中染色10 min,洗滌后用刀片小心割下膜，置于載玻片上，用中性樹(shù)膠封片；在顯微鏡下，隨機(jī)選取5個(gè)視野(100×)，計(jì)數(shù)穿膜細(xì)胞個(gè)數(shù)。

1.5 化療藥物藥敏試驗(yàn) 分別取對(duì)數(shù)生長(zhǎng)期的AGS/FoxP3和AGS/vector細(xì)胞，計(jì)數(shù)，接種至96孔板，每孔5 000個(gè)細(xì)胞。每組設(shè)5個(gè)復(fù)孔。細(xì)胞貼壁8 h后，分別加入不同濃度的5-FU或伊立替康；48 h后用MTT法在550 nm波長(zhǎng)處檢測(cè)D值。

1.6 統(tǒng)計(jì)學(xué)處理 采用SPSS 17.0進(jìn)行統(tǒng)計(jì)分析。滿足正態(tài)性分布和方差齊性的兩組間比較采用t檢驗(yàn)，不滿足者采用非參數(shù)Kruskal-Wallis檢驗(yàn)。檢驗(yàn)水準(zhǔn)(α)為0.05。

2 結(jié) 果

2.1 單克隆挑選及過(guò)表達(dá)載體的鑒定 經(jīng)Western 印跡驗(yàn)證，選取蛋白表達(dá)最強(qiáng)的5號(hào)克隆(圖1)擴(kuò)大培養(yǎng)用于后續(xù)試驗(yàn)。

圖1 篩選FoxP3蛋白過(guò)表達(dá)最強(qiáng)克隆

2.2 流式細(xì)胞術(shù)鑒定細(xì)胞轉(zhuǎn)染效率 經(jīng)過(guò) G418篩選和單克隆挑選擴(kuò)增的穩(wěn)定轉(zhuǎn)染的AGS細(xì)胞(圖2A)，熒光顯微鏡下見(jiàn)細(xì)胞均帶有綠色熒光(圖2B)，說(shuō)明轉(zhuǎn)染成功，流式細(xì)胞技術(shù)顯示轉(zhuǎn)染效率近100%(圖2C)。

2.3 過(guò)表達(dá)FoxP3對(duì)胃癌細(xì)胞增殖能力的影響 細(xì)胞增殖實(shí)驗(yàn)均顯示轉(zhuǎn)染FoxP3質(zhì)粒后，AGS細(xì)胞的增殖能力減低(圖3)，提示AGS/FoxP3具有抑制胃癌細(xì)胞增殖的作用(P<0.05)。

圖2 FoxP3穩(wěn)定轉(zhuǎn)染細(xì)胞系的篩選

圖3 MTT示AGS/FoxP3抑制AGS細(xì)胞生長(zhǎng)

2.4 細(xì)胞周期的變化 流式細(xì)胞術(shù)分析結(jié)果(圖4)表明：轉(zhuǎn)染FoxP3后，細(xì)胞被阻滯在G1期，G1期細(xì)胞所占百分比明顯多于未轉(zhuǎn)染vector的對(duì)照組[G1期：(85.21±10.21)%vs(72.12±7.34)%,P<0.05]。

圖4 流式細(xì)胞術(shù)提示細(xì)胞周期被阻滯在G1期

2.5 轉(zhuǎn)染FoxP3對(duì)胃癌細(xì)胞遷移能力的影響 細(xì)胞遷移實(shí)驗(yàn)(圖5)表明：與對(duì)照組相比，轉(zhuǎn)染FoxP3的AGS細(xì)胞在高倍視野中平均穿膜細(xì)胞數(shù)明顯減少[(203±42)個(gè)/HPvs(891±100)個(gè)/HP,P<0.05]，說(shuō)明轉(zhuǎn)染FoxP3后胃癌細(xì)胞的遷移能力減弱。

圖5 轉(zhuǎn)染FoxP3胃癌細(xì)胞的遷移能力減弱

2.6 轉(zhuǎn)染后FoxP3對(duì)胃癌細(xì)胞化療敏感性的影響 在5-FU 100 μg/mL和500 μg/mL濃度下，轉(zhuǎn)染FoxP3的AGS細(xì)胞生長(zhǎng)抑制率明顯高于對(duì)照組；在伊立替康25 μg/mL及50 μg/mL藥物濃度下，轉(zhuǎn)染FoxP3基因的AGS細(xì)胞的生長(zhǎng)抑制率明顯升高(P<0.05)。結(jié)果表明，F(xiàn)oxP3可以增加胃癌細(xì)胞對(duì)化療藥物的敏感性(圖6)。

圖6 化療藥物敏感性的增加

3 討 論

FoxP3對(duì)Treg細(xì)胞的形成和功能起關(guān)鍵作用[9]。FoxP3陽(yáng)性的T細(xì)胞大部分為T(mén)reg，它們是構(gòu)成腫瘤免疫的重要因素。有研究[10]報(bào)道，胰腺癌新輔助放化療后腫瘤微環(huán)境中Treg減少及相關(guān)的細(xì)胞毒作用減弱。放療可使循環(huán)中的Treg明顯下降，患者無(wú)瘤生存時(shí)間延長(zhǎng)[11-12]。但是，有證據(jù)[13]發(fā)現(xiàn)，Treg比細(xì)胞毒性T淋巴細(xì)胞(cytotoxic T lymphocyte, CTL) 更耐受離子照射，放療后局部原位組織中Treg不減少是致放療耐受的主要原因。在動(dòng)物模型中也發(fā)現(xiàn),Treg比其他淋巴細(xì)胞更耐受放療，他們可能在放療相關(guān)的組織損傷中有自穩(wěn)態(tài)作用，導(dǎo)致放療不敏感[14]。與FoxP3相互作用的蛋白質(zhì)中有視黃酸受體孤兒受體樣受體α(ROR-α)，F(xiàn)oxP3可以抑制ROR-α介導(dǎo)的轉(zhuǎn)錄激活作用[15]。當(dāng)缺乏配體時(shí)，ROR-α可以與轉(zhuǎn)錄共抑制因子結(jié)合；當(dāng)與配體結(jié)合后，ROR-α發(fā)生形變與共活化因子相結(jié)合[16]，從而調(diào)節(jié)其轉(zhuǎn)錄抑制或活化作用。

近來(lái)發(fā)現(xiàn)多種腫瘤細(xì)胞表達(dá)FoxP3，且FoxP3與淋巴結(jié)轉(zhuǎn)移和預(yù)后有重要關(guān)系[17-19]。在亞洲胃癌患者中，腫瘤組織中浸潤(rùn)的CD3、CD8、CD45RO、FoxP3等免疫分子的表達(dá)與預(yù)后有關(guān)，說(shuō)明免疫狀態(tài)水平可影響患者預(yù)后[20]。既往研究多集中于Treg在腫瘤免疫中的作用，而忽視了腫瘤細(xì)胞表達(dá)FoxP3。腫瘤細(xì)胞表達(dá)的FoxP3可通過(guò)影響T淋巴細(xì)胞的分化而影響腫瘤免疫應(yīng)答，如結(jié)腸癌細(xì)胞中，F(xiàn)oxP3與Treg發(fā)生相互作用，影響幼稚T淋巴細(xì)胞的分化，從而影響免疫效應(yīng)[21]；肝癌細(xì)胞可通過(guò)分泌轉(zhuǎn)化生長(zhǎng)因子β1(TGF-β1)誘導(dǎo)淋巴細(xì)胞中FoxP3的表達(dá)，從而抑制其抗腫瘤免疫反應(yīng)[22]。我們的前期研究[23]也發(fā)現(xiàn),將胃癌細(xì)胞與外周單個(gè)核淋巴細(xì)胞(PBMC)共培養(yǎng)后,FoxP3增多;而Treg也通過(guò)分泌抑制性細(xì)胞因子抑制胃癌細(xì)胞的生長(zhǎng)。我們前期研究[24]發(fā)現(xiàn),胃癌組織中胃癌細(xì)胞和淋巴細(xì)胞中均表達(dá)FoxP3，而且胃癌細(xì)胞FoxP3的表達(dá)與預(yù)后良好有關(guān)，而高密度浸潤(rùn)的Treg與預(yù)后差有關(guān)，風(fēng)險(xiǎn)模型分析提示Treg是影響預(yù)后的主要因素。然而，F(xiàn)oxP3基因?qū)ξ赴┘?xì)胞具體生物學(xué)行為的影響目前還不清楚。

乳腺癌體內(nèi)外實(shí)驗(yàn)[25-27]均表明,FoxP3能抑制乳腺癌的生長(zhǎng)。乳腺癌細(xì)胞系過(guò)表達(dá)FoxP3基因后，細(xì)胞增殖、遷移和侵襲能力均降低[25]。FoxP3可通過(guò)抑制人表皮生長(zhǎng)因子受體2(HER2)和S相激酶相關(guān)蛋白2(SKP2)的轉(zhuǎn)錄活性和誘導(dǎo)腫瘤抑制基因p21的活性抑制乳腺癌的生長(zhǎng)[28-29]。前列腺癌細(xì)胞系增加FoxP3表達(dá)后生長(zhǎng)明顯抑制。另外敲除FoxP3會(huì)導(dǎo)致早發(fā)的前列腺增生和上皮內(nèi)瘤變[30]。本實(shí)驗(yàn)發(fā)現(xiàn)過(guò)表達(dá)FoxP3的胃癌細(xì)胞增殖減慢、遷移能力減弱，胃癌細(xì)胞主要被阻滯在G1期。我們以往研究[31]還發(fā)現(xiàn)，F(xiàn)oxP3促進(jìn)胃癌細(xì)胞的凋亡與Caspase相關(guān)的凋亡信號(hào)通路有關(guān)，因此推測(cè)FoxP3在胃癌中可能起抑癌作用。本研究發(fā)現(xiàn)，轉(zhuǎn)染FoxP3的胃癌細(xì)胞對(duì)5-FU和伊立替康兩種化療藥物的敏感性增加，這可能也是FoxP3影響患者預(yù)后的原因之一。Treg的免疫抑制作用是影響預(yù)后的重要因素，而胃癌細(xì)胞中FoxP3起抑癌作用，它們可能共同參與調(diào)節(jié)腫瘤微環(huán)境的狀態(tài)，最終決定患者預(yù)后。

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[本文編輯] 姬靜芳

The role and mechanism of FoxP3 in biological behavior of gastric cancer cells

MA Gui-fen1, PAN Du-yi2, HE Jian1, ZENG Zhao-chong1, CHEN Shi-yao2*

1. Department of Radiotherapy, Zhongshan Hospital, Fudan University, Shanghai 200032, China 2. Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai 200032, China

Objective： To explore the role and mechanism of Forkhead box protein 3 (FoxP3) in biological behavior of gastric cancer (GC) cells.Methods：Invitro, the plasmid of FoxP3-shRNA was transfected into GC cells, and then the stably transfected cells were established by drug-screening and monoclone-selection. MTT assay was used to detect the growth and proliferation of GC cells. And cell cycle was detected by flow cytometry. MTT assay was used to measure the difference regarding sensitivity to chemotherapy drugs.Results： GC cell line which was stably transfected with FoxP3 gene, was established. GC cell line with up-regulation of FoxP3 gene, compared to the vector-transfected control, showed slower growth and proliferation rate, weaker ability of invasion (transmembrane cell counts: [203±42] cells/HPvs[891±100] cells/HP,P<0.05), and higher sensitivity to chemotherapy drugs(P<0.05).Conclusions： FoxP3 gene plays a role in inhibiting the growth of GC cells.

Forkhead box protein; gastric cancer; gene function; biological behavior

2015-12-22 [接受日期] 2016-05-19

國(guó)家自然科學(xué)基金青年科學(xué)基金項(xiàng)目(81502005)，上海市自然科學(xué)基金(14ZR1406600). Supported by Youth Program of National Natural Science Foundation of China (8150200) and Natural Science Foundation of Shanghai (14ZR1406600).

馬桂芬，碩士，住院醫(yī)師. E-mail:ma.guifen@zs-hospital.sh.cn

*通信作者(Corresponding author). Tel: 021-64041990, E-mail: chen.shiyao@zs-hospital.sh.cn

10.12025/j.issn.1008-6358.2016.20160931

R 735.2

A