史惠蓉 吳開(kāi)元 張海玲 劉惠娜

鄭州大學(xué)第一附屬醫(yī)院婦產(chǎn)科，河南 鄭州 450052

DLC1基因?qū)θ寺殉舶┘?xì)胞OVCAR-3順鉑耐藥性的影響

史惠蓉 吳開(kāi)元 張海玲 劉惠娜

鄭州大學(xué)第一附屬醫(yī)院婦產(chǎn)科，河南 鄭州 450052

背景與目的：有研究發(fā)現(xiàn)，肝癌缺失基因1(DLC1)在多種腫瘤中低表達(dá)或不表達(dá)，其通過(guò)調(diào)節(jié)黏著斑激酶(FAK)、促分裂原活化蛋白激酶(MAPK)等影響腫瘤細(xì)胞的凋亡。而對(duì)于DLC1在卵巢癌中的表達(dá)及作用等的研究甚少，本實(shí)驗(yàn)采用DLC1基因轉(zhuǎn)染該基因表達(dá)缺失的人卵巢癌多藥耐藥細(xì)胞系OVCAR-3，觀察轉(zhuǎn)染前后該細(xì)胞對(duì)順鉑耐藥性及FAK、p38MAPK的變化。方法：將OVCAR-3細(xì)胞分為3組，空白組為未經(jīng)處理的OVCAR-3細(xì)胞，陰性對(duì)照組轉(zhuǎn)染空質(zhì)粒pEGFP-C3，實(shí)驗(yàn)組轉(zhuǎn)染重組質(zhì)粒pEGFP-C3-DLC1。RT-PCR和Western blot檢測(cè)各組細(xì)胞中DLC1基因和蛋白的表達(dá)，四甲基偶氮唑藍(lán)(MTT)法測(cè)定各組細(xì)胞對(duì)順鉑的半數(shù)抑制濃度(IC50)，Western blot觀察FAK、p38蛋白及其磷酸化水平的變化，流式細(xì)胞儀測(cè)定順鉑處理前后各組細(xì)胞凋亡率及周期分布改變。結(jié)果：DLC1基因和蛋白在實(shí)驗(yàn)組表達(dá)而空白對(duì)照組和陰性對(duì)照組均未見(jiàn)表達(dá)，實(shí)驗(yàn)組細(xì)胞與其他兩組細(xì)胞相比，對(duì)順鉑的IC50較低(4.02vs4.99/4.90 μmol/L，P＜0.01)，p-p38蛋白表達(dá)上升而p-FAK蛋白表達(dá)下降(3.02vs1.52/1.61，3.13vs9.03/8.99，P＜0.01)，順鉑處理前后實(shí)驗(yàn)組與其他兩組細(xì)胞相比凋亡率增加(8.97%vs1.81%/1.95%，30.68%vs18.03%/20.33%，P＜0.01)，G1期細(xì)胞比例增加(65.80%vs60.82%/59.80%，66.48%vs55.42%/53.94%，P＜0.05)。結(jié)論：轉(zhuǎn)染DLC1基因可使OVCAR-3G1期細(xì)胞比例及凋亡率升高，對(duì)順鉑敏感性增加，該作用可能依賴(lài)于細(xì)胞內(nèi)外源性DLC1基因的表達(dá)、p-FAK表達(dá)降低和p-p38表達(dá)增強(qiáng)。

DLC1基因； 卵巢癌； 逆轉(zhuǎn)耐藥； 凋亡； 細(xì)胞周期

卵巢癌治療過(guò)程中易出現(xiàn)耐藥和復(fù)發(fā)，其耐藥性產(chǎn)生的原因之一是腫瘤細(xì)胞凋亡途徑如PI3K/AKT、MAPK和Jak/STAT等信號(hào)通路的異常，黏著斑激酶(FAK)是這些信號(hào)通路的交匯點(diǎn)［1-2］。肝癌缺失基因1(DLC1)可調(diào)控黏著斑蛋白去磷酸化，激活p38MAPK家族，進(jìn)而介導(dǎo)順鉑引起的卵巢癌細(xì)胞的凋亡［3-4］。由此推測(cè)，DLC1基因可作為上游調(diào)控基因調(diào)節(jié)卵巢癌細(xì)胞的凋亡，通過(guò)轉(zhuǎn)染使不表達(dá)DLC1基因的卵巢癌順鉑耐藥細(xì)胞重新表達(dá)外源性DLC1基因，可促進(jìn)癌細(xì)胞凋亡，達(dá)到逆轉(zhuǎn)耐藥的目的。DLC1基因在多種腫瘤的生物學(xué)行為中具有重要的作用，本課題前期研究發(fā)現(xiàn)DLC1的低表達(dá)或表達(dá)缺失與卵巢上皮性癌的發(fā)生、發(fā)展有關(guān)［5］。本階段實(shí)驗(yàn)采用重組質(zhì)粒pEGFP-C3-DLC1轉(zhuǎn)染不表達(dá)DLC1基因的卵巢癌多藥耐藥細(xì)胞OVCAR-3［6］，通過(guò)檢測(cè)相關(guān)蛋白的變化及細(xì)胞周期分布、凋亡率的變化，初步探討DLC1基因?qū)β殉舶┘?xì)胞凋亡和耐藥的影響及可能的作用機(jī)制。

1 材料和方法

1.1 藥物和試劑

pEGFP-C3-DLC1質(zhì)粒由美國(guó)國(guó)立癌癥研究所Popescu NC教授惠贈(zèng)［7］；卵巢癌多藥耐藥細(xì)胞系 OVCAR-3購(gòu)自中科院腫瘤研究所細(xì)胞中心，OVCAR-3細(xì)胞系由耐藥卵巢癌患者腹水中癌細(xì)胞體外培養(yǎng)建系，不表達(dá)DLC1［6］，是一種對(duì)順鉑、多柔比星和環(huán)磷酰胺等化療藥物耐藥的細(xì)胞系；順鉑(DDP，山東齊魯制藥廠(chǎng))；PCR引物(上海生工公司)；LipofectamineTM2000陽(yáng)離子脂質(zhì)體轉(zhuǎn)染試劑(美國(guó)Invitrogen公司)；兔抗人DLC1(H-260)抗體、鼠抗人FAK抗體(美國(guó)Santa Cruz公司)；兔抗β-actin抗體(北京博奧森公司)；兔抗人磷酸化FAK(p-FAK)抗體(Tyr397)、兔抗人p38和磷酸化p38(p-p38)(Tyr180/182)抗體(美國(guó)Bioworld公司)；Annexin V-FITC細(xì)胞凋亡檢測(cè)試劑盒(江蘇碧云天公司)。

1.2 質(zhì)粒的提取、擴(kuò)增

pEGFP-C3-DLC1質(zhì)粒DNA轉(zhuǎn)化感受態(tài)JM109菌株，LB培養(yǎng)基擴(kuò)增，無(wú)內(nèi)毒素質(zhì)粒小量提取試劑盒提取質(zhì)粒DNA，限制性?xún)?nèi)切酶BamHⅠ及EcoRⅠ雙酶切鑒定。

1.3 細(xì)胞培養(yǎng)、轉(zhuǎn)染

OVCAR-3細(xì)胞用含10%胎牛血清的DMEM高糖培養(yǎng)基(含10 μg/mL青霉素及10 μg/mL鏈霉素)置37 ℃、CO2體積分?jǐn)?shù)為5%的培養(yǎng)箱中培養(yǎng)，并分為3組：空白組為未處理的OVCAR-3細(xì)胞，陰性對(duì)照組細(xì)胞轉(zhuǎn)染質(zhì)粒pEGFP-C3，實(shí)驗(yàn)組轉(zhuǎn)染重組質(zhì)粒pEGFP-C3-DLC1。按脂質(zhì)體轉(zhuǎn)染說(shuō)明書(shū)進(jìn)行轉(zhuǎn)染，其中DNA和脂質(zhì)體比例為1 μg∶1.5 μL，G418維持篩選濃度為800 μg/mL。篩選維持21 d，至轉(zhuǎn)染組出現(xiàn)明顯的陽(yáng)性細(xì)胞克隆。 熒光顯微鏡下觀察轉(zhuǎn)染前后各組細(xì)胞形態(tài)的變化。

1.4 RT-PCR檢測(cè)DLC1 mRNA的表達(dá)

TRIzol分別提取3組細(xì)胞總RNA，逆轉(zhuǎn)錄合成cDNA第一鏈，以其為模板PCR擴(kuò)增DLC1 cDNA。DLC1基因引物上游序列(產(chǎn)物片段為471 bp)：5’-TTCTGATGAGGGAGATTCGG-3’，下游序列：5’-ACGTTGACCGTCAGTGGGAC-3’；β-actin引物上游序列(產(chǎn)物片段為372bp)：5’-ACAATGAGCTGCGTGTGGCT-3’，下游序列：5’-TCTCCTTAATGTCACGCACGA-3’。PCR反應(yīng)條件為：94 ℃預(yù)變性3 min；94 ℃變性30 s，56.5 ℃退火30 s，72 ℃延伸30 s，30次循環(huán)；72 ℃終延伸5 min，2×Taq Mix 12.5 μL，上下游引物各1 μL，模版cDNA 1 μL，無(wú)RNase水補(bǔ)充至總體積25 μL，PCR反應(yīng)產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳，結(jié)果用Gene Genius Bio Imaging System成像分析。

1.5 Western blot方法［8］測(cè)定DLC1、FAK、p-FAK、p38和p-p38蛋白的表達(dá)

分別提取3組細(xì)胞的總蛋白，進(jìn)行SDSPAGE凝膠電泳，轉(zhuǎn)膜，封閉，一抗4 ℃溫育過(guò)夜，HRP標(biāo)記的二抗室溫溫育1 h，ECL化學(xué)發(fā)光、曝光。結(jié)果用BANDSCAN軟件進(jìn)行分析，計(jì)算目的條帶與內(nèi)參(β-actin)的灰度值比值。

1.6 MTT法測(cè)定DLC1基因?qū)?xì)胞增殖的影響和藥物對(duì)各組細(xì)胞的毒性作用(IC50)

本實(shí)驗(yàn)所用順鉑的濃度梯度為2.5、5、10、20、40 μmol/L，加藥培養(yǎng)48 h后進(jìn)行MTT法分析，測(cè)定A492nm分光光度值，計(jì)算不同濃度順鉑對(duì)3組細(xì)胞的抑制率和3組細(xì)胞對(duì)順鉑的IC50。抑制率=(A陰性對(duì)照均值-A加藥孔均值)/A陰性對(duì)照均值。IC50由計(jì)算軟件測(cè)算。在順鉑用量均為空白組細(xì)胞的IC50值時(shí)，測(cè)定各組細(xì)胞的凋亡率及周期分布。

1.7 統(tǒng)計(jì)學(xué)處理

應(yīng)用SPSS 16.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析，結(jié)果以表示，多組定量資料均數(shù)的比較采用單因素方差分析(one-way ANOVA)，以α=0.05為檢驗(yàn)水準(zhǔn)，組間差異檢驗(yàn)采用bonferroni法，以α=0.01為檢驗(yàn)水準(zhǔn)。順鉑處理前后各組細(xì)胞相關(guān)指標(biāo)的比較采用配對(duì)t檢驗(yàn)，以α=0.05為檢驗(yàn)水準(zhǔn)。p-FAK、p-p38與各組細(xì)胞對(duì)順鉑IC50的關(guān)系行Pearson相關(guān)性分析。

2 結(jié) 果

2.1 質(zhì)粒的雙酶切鑒定

限制性?xún)?nèi)切酶BamHⅠ及EcoRⅠ雙酶切鑒定重組質(zhì)粒pEGFP-C3-DLC1 DNA，電泳后出現(xiàn)4.7×103bp和3.9×103bp兩條清晰條帶，分別為pEGFP-C3質(zhì)粒和DLC1 cDNA片段(圖1)。

2.2 熒光顯微鏡下觀察轉(zhuǎn)染后3組細(xì)胞

空白組細(xì)胞未見(jiàn)綠色熒光蛋白表達(dá)，陰性對(duì)照組和實(shí)驗(yàn)組細(xì)胞均可見(jiàn)綠色熒光蛋白表達(dá)，證明轉(zhuǎn)染成功(圖2)。

2.3 3組細(xì)胞中DLC1基因的表達(dá)差異

DLC1基因擴(kuò)增產(chǎn)物片段在實(shí)驗(yàn)組清晰可見(jiàn)，而在空白組和陰性對(duì)照組均未見(jiàn)表達(dá)(圖3)。

2.4 DLC1、FAK、p-FAK、p38和p-p38蛋白在3組細(xì)胞中的表達(dá)變化

DLC1蛋白在實(shí)驗(yàn)組可見(jiàn)陽(yáng)性表達(dá)而在空白組和陰性對(duì)照組均未見(jiàn)表達(dá)，實(shí)驗(yàn)組與其他兩組相比：p-FAK表達(dá)水平降低(F=303 192.7，P＜0.01)，p-p38表達(dá)水平升高(F=18 646.88，P＜0.01)，但總FAK和p-p38在3組細(xì)胞中的表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(F=0.01，P=1.00；F=0.26，P=0.78)(表1、圖4)。

表 1 DLC1對(duì)FAK和p38磷酸化水平的影響Tab. 1 The effect of DLC1 gene on the phosphorylation level of FAK and p38(10-1)

2.5 不同濃度順鉑對(duì)3組細(xì)胞的抑制率和IC50

相同順鉑濃度下實(shí)驗(yàn)組細(xì)胞的抑制率明顯高于陰性對(duì)照組和空白組(圖5)。實(shí)驗(yàn)組細(xì)胞對(duì)順鉑的IC50為(4.02±0.18)μmol/L較空白組(4.99±0.09)μmol/L和陰性對(duì)照組(4.90±0.29)μmol/L顯著減少(F=35.27，P＜0.01)。

2.6 細(xì)胞凋亡率和周期的變化

順鉑處理前后實(shí)驗(yàn)組細(xì)胞的凋亡率高于空白組和陰性對(duì)照組(F=65 509.33，P＜0.01；F=3 888.74，P＜0.01，表2、圖6)，順鉑處理后空白組和陰性對(duì)照組G2期細(xì)胞明顯高于加藥前(t=-4.35，P=0.01；t=-6.01，P＜0.01)，而實(shí)驗(yàn)組G1期細(xì)胞比例顯著高于加藥前(t=-2.93，P=0.04)，加藥前后實(shí)驗(yàn)組G1期細(xì)胞比例顯著高于其他兩組(F=35.49，P＜0.01；F=298.86，P＜0.01，表3、圖7)。

他們都是獲得非凡成就的杰出人物，成功的關(guān)鍵就是為自己偷來(lái)了寶貴的時(shí)間，而時(shí)間就是生命，時(shí)間充裕了，干事業(yè)的機(jī)會(huì)和空間也大大增加了，再加上合適的方法和正確的目標(biāo)，人生成功的大門(mén)自然也就不難推開(kāi)了。

2.7 p-FAK和p-p38蛋白的表達(dá)變化與3組細(xì)胞對(duì)順鉑IC50值的相關(guān)性分析

p-FAK的表達(dá)水平與3組細(xì)胞對(duì)順鉑的IC50呈正相關(guān)(r=0.89，P＜0.01)，p-p38蛋白的表達(dá)水平與3組細(xì)胞對(duì)順鉑的IC50呈負(fù)相關(guān)(r=-0.90，P＜0.01)。

3 討 論

DLC1在正常卵巢組織中陽(yáng)性表達(dá)，在卵巢癌細(xì)胞中不表達(dá)或低表達(dá)［9］，是Yuan等［10］1998年在肝癌組織中發(fā)現(xiàn)的抑癌基因，定位于人類(lèi)染色體8p21.3-22，編碼相對(duì)分子質(zhì)量為123×103的蛋白，通過(guò)負(fù)性調(diào)控Rho蛋白及其效應(yīng)因子和調(diào)控黏著斑蛋白包括FAK去磷酸化發(fā)揮其抑制腫瘤形成、增殖，誘導(dǎo)腫瘤細(xì)胞凋亡的生物學(xué)功能［3］。

表 2 順鉑和DLC1基因?qū)VCAR-3細(xì)胞凋亡率(%)的影響Tab. 2 The effect of cisplatin and DLC1 gene on the apoptotic ratio(%) of OVCAR-3 cells

表 3 順鉑和DLC1基因?qū)VCAR-3細(xì)胞細(xì)胞周期的影響Tab. 3 The effect of cisplatin and DLC1 gene on the cell cycle of OVCAR-3 cells

DLC1使OVCAR-3細(xì)胞G1期比例和早期凋亡率均增加，而順鉑使OVCAR-3細(xì)胞阻滯于G2期［11］，與DLC1同時(shí)作用時(shí)，能使癌細(xì)胞更多地停滯于DNA合成前期，抑制腫瘤細(xì)胞的增殖、分裂，提高癌細(xì)胞的早期凋亡率。這種對(duì)細(xì)胞周期的影響與在腎癌細(xì)胞中相似，DLC1可通過(guò)調(diào)節(jié)腎癌細(xì)胞周期蛋白使癌細(xì)胞阻滯于G0/G1期［12］，抑制腎癌細(xì)胞的生長(zhǎng)。DLC1促進(jìn)OVCAR-3凋亡的作用，一方面可能與DLC1能夠使p-FAK去磷酸化而失活，細(xì)胞失去基質(zhì)缺少粘附，發(fā)生失巢凋亡有關(guān)［13］，表現(xiàn)為DLC1單獨(dú)作用時(shí)細(xì)胞的早期凋亡率顯著增加；另一方面則可能通過(guò)調(diào)控黏著斑蛋白去磷酸化，激活p38MAPK家族，進(jìn)而介導(dǎo)順鉑引起的卵巢癌細(xì)胞的凋亡［3-4］，表現(xiàn)為OVCAR-3細(xì)胞在順鉑和外源性DLC1基因的共同作用下較只受外源性DLC1基因影響時(shí)早期凋亡率增加，另OVCAR-3細(xì)胞在順鉑作用時(shí)，攜帶外源性DLC1基因的OVCAR-3細(xì)胞較無(wú)外源性DLC1基因的OVCAR-3早期凋亡率增加，這說(shuō)明DLC1的低表達(dá)或表達(dá)缺失可能是卵巢癌細(xì)胞無(wú)限增殖、凋亡受限和化療耐藥的原因之一。

順鉑引起卵巢癌細(xì)胞的凋亡大多與p38MAPK的激活有關(guān)［4］。Villedieu等［14］認(rèn)為卵巢癌順鉑化療過(guò)程中，P38活性減低和FAK活性持續(xù)高表達(dá)與卵巢癌細(xì)胞順鉑耐藥性的產(chǎn)生相關(guān)。本研究發(fā)現(xiàn)轉(zhuǎn)染DLC1基因后OVCAR-3細(xì)胞對(duì)順鉑的IC50降低，敏感性增強(qiáng)，與p-FAK的表達(dá)呈負(fù)相關(guān)，與p-p38的表達(dá)呈正相關(guān)，其中FAK和p38活性的變化與卵巢癌對(duì)順鉑耐藥性之間的關(guān)系與前學(xué)者的研究結(jié)果一致，進(jìn)一步發(fā)現(xiàn)了DLC1基因可能是調(diào)控這一逆轉(zhuǎn)耐藥過(guò)程的上游基因之一，為發(fā)現(xiàn)新的卵巢癌生物治療靶點(diǎn)提供理論依據(jù)。

綜上所述，DLC1可能作用于G1/S檢查點(diǎn)使癌細(xì)胞阻滯于G1期，該基因的表達(dá)繼而使FAK失活、p38激活，增加卵巢癌細(xì)胞對(duì)順鉑的敏感性，起到一定程度逆轉(zhuǎn)耐藥的作用。本研究首次在腫瘤中探討了DLC1與腫瘤耐藥的關(guān)系，為下一步建立動(dòng)物耐藥模型，進(jìn)行逆轉(zhuǎn)耐藥的體內(nèi)實(shí)驗(yàn)研究奠定了基礎(chǔ)。當(dāng)然，本研究?jī)H限于卵巢癌耐藥與DLC1的關(guān)系，提示其很可能是逆轉(zhuǎn)卵巢癌順鉑耐藥的一個(gè)候選靶點(diǎn)，該通路的具體調(diào)控機(jī)制和各分子間的因果關(guān)系仍需進(jìn)一步研究來(lái)證實(shí)。

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The effect of DLC1 gene on chemoresistance in human ovarian cancer cell line OVCAR-3

SHI Huirong,WU Kai-yuan,ZHANG Hai-ling,LIU Hui-na(Department of Gynecology and Obstetrics,the First Affiliated Hospital, Zhengzhou University,Zhengzhou Henan 450052, China)

SHI Hui-rong E-mail：huirongshi@yahoo.com.cn

Background and purpose：The deleted in liver cancer gene1 (DLC1) is lowly or negatively expressed in different cancers and have an effect on the apoptosis of cancer cells due to the regulation of focal adhesion kinase (FAK) and mitogen-actived protein kinase (MAPK). However, the expression level and role of the DLC1 gene in ovarian cancer has been rarely studied. This study aimed to transfect the DLC1 gene into the OVCAR-3 cells, where the DLC1 gene was once negatively expressed, so as to investigate the effect of the DLC1 gene on chemoresistance as well as a variation of FAK and p38MAPK.Methods：Ovarian cancer cell line OVCAR-3 was treated with none(blank group), empty plasmid pEGFP-C3(negative control group) and pEGFP-C3-DLC1(experimental group). The expression of DLC1 mRNA and protein were detected separately using RT-PCR and Western blot. The IC50 of cisplatin was determined using the Methyl thiazolyl tetrazolium test (MTT). The changes of FAK and p38 protein expression and their phosphorylated status were determined by Western blot. Apoptosis and cell cycle distribution were detected by fl ow cytometry.Results：DLC1 gene and protein were only observed in the experiment group rather than in the other groups. The IC50 of the experiment group was lower than in the other groups (4.02vs4.99/4.90 μmol/L,P＜0.01).There was a higher expression of p-p38 but lower expression of p-FAK (3.02vs1.52/1.61, 3.13vs9.03/8.99,P＜0.01)after the treatment of cisplatin in experimental group. Apoptosis was significantly higher and G1 arrest was oberved only in the experimental group after the treatment of cisplatin, there were statistically differences among the groups(P＜0.01).Conclusion：The OVCAR-3 cells were more sensitive to cisplatin after transient transfection of DLC1 gene in terms of the induction of apoptosis and G1 arrest, which might be through the upregulation of p-FAK expression and downregulation of p-p38 expression.

DLC1 gene; Ovarian cancer; Reverse chemoresistance; Apoptosis; Cell cycle

10.3969/j.issn.1007-3969.2011.02.005

R737.31；R73-36+1

A

1007-3639(2011)02-0103-07

河南省醫(yī)學(xué)科技攻關(guān)計(jì)劃重大項(xiàng)目(No:20090113)。

史惠蓉 E-mail:huirongshi@yahoo.com.cn

2010-11-01

2011-01-XX）