·論著·

沉默S100A4基因表達(dá)對(duì)鼻咽癌細(xì)胞株CNE2凋亡及侵襲的影響

羅海林1，江青山1，沈?qū)氒?，楊學(xué)敏2

(1南華大學(xué)附屬第一醫(yī)院，湖南衡陽(yáng)421001；2廣西醫(yī)科大學(xué)第一附屬醫(yī)院)

摘要：目的觀察沉默鈣離子相關(guān)蛋白(S100A4)表達(dá)對(duì)鼻咽癌細(xì)胞株CNE2凋亡及侵襲的影響。方法培養(yǎng)CNE2細(xì)胞，分為空白組、對(duì)照組及實(shí)驗(yàn)組。實(shí)驗(yàn)組細(xì)胞通過(guò)Lipofectamine2000轉(zhuǎn)染S100A4 siRNA，對(duì)照組細(xì)胞通過(guò)Lipofectamine2000轉(zhuǎn)染空質(zhì)粒，空白組細(xì)胞未做轉(zhuǎn)染。Western blotting法檢測(cè)CNE2細(xì)胞中的S100A4蛋白。real-time PCR法檢測(cè)S100A4 mRNA。流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡情況。Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲能力。結(jié)果實(shí)驗(yàn)組、空白組、對(duì)照組S100A4 mRNA相對(duì)表達(dá)量分別為0.600 4±0.05、1.000 0±0.00、0.894 1±0.09，S100A4 蛋白相對(duì)表達(dá)量分別為0.22±0.016、0.42±0.022、0.39±0.022。實(shí)驗(yàn)組細(xì)胞中S100A4 mRNA、蛋白表達(dá)量與空白組及對(duì)照組相比，P均<0.05。實(shí)驗(yàn)組、空白組、對(duì)照組細(xì)胞凋亡率分別為45.87%、3.49%、2.49%，實(shí)驗(yàn)組與空白組及對(duì)照組相比，P均<0.05。Transwell實(shí)驗(yàn)結(jié)果示實(shí)驗(yàn)組穿膜細(xì)胞數(shù)為(206±22)個(gè)，空白組和對(duì)照組分別為(329±12)、(347±21)個(gè)。實(shí)驗(yàn)組穿膜細(xì)胞數(shù)與空白組、對(duì)照組相比，P均<0.05。結(jié)論 沉默S100A4基因表達(dá)后，CNE2細(xì)胞凋亡增多，細(xì)胞侵襲能力漸弱。

關(guān)鍵詞：鼻咽腫瘤；鼻咽癌；鈣離子相關(guān)蛋白；S100A4蛋白；細(xì)胞凋亡；細(xì)胞侵襲

doi：10.3969/j.issn.1002-266X.2015.39.001

中圖分類號(hào)：R739．63文獻(xiàn)標(biāo)志碼：A

基金項(xiàng)目：湖南省科技廳科研基金資助項(xiàng)目(2012SK3159)。

作者簡(jiǎn)介：第一羅海林(1990-)，男，碩士研究生，研究方向?yàn)槎茄屎眍^頸腫瘤。E-mail: 455155631@qq.com

作者簡(jiǎn)介：通信江青山(1971-)，男，碩士研究生，副主任醫(yī)師，副教授，碩士生導(dǎo)師，主要研究方向?yàn)槎呛砜萍膊〉脑\治。E-mail: 2365486131@qq.com

收稿日期：(2015-08-23)

Effect of silence of S100A4 gene on apoptosis and invasion

of nasopharyngeal carcinoma cell line CNE2

LUOHai-lin1,JIANGQing-shan,SHENBao-ming,YANGXue-min

(1TheFirstAffiliatedHospitalofUniversityofSouthChina,Hengyang421001,China)

Abstract:ObjectiveTo observe the effect of silencing calcium ion associated protein (S100A4) expression on apoptosis and invasion of nasopharyngeal carcinoma cell line CNE2. Methods The CNE2 cells were cultured, and then were divided into the blank group, control group and experimental group. The cells of the experimental group were transfected with S100A4siRNA by Lipofectamine2000, the control group was transfected with CON (empty plasmid) by Lipofectamine2000, and the cells in the blank group did not receive special transfection. Western blotting was used to detect the S100A4 protein in CNE2 cells, and the expression of S100A4 mRNA was detected by real-time PCR. The apoptosis was detected by flow cytometry. Transwell assay was used to detect cell invasion. ResultsThe relative expression levels of S100A4 mRNA in the experimental group, blank control group and control group were respectively 0.600 4±0.05, 1.000 0±0.00 and 0.894 1±0.09, and the relative expression levels of S100A4 protein were respectively 0.22±0.016, 0.42±0.022 and 0.39±0.022. The expression of S100A4 mRNA and protein in the experimental group was lower than that in the control group and the blank control group (all P<0.05). The apoptosis rates of the experimental group, blank group and control group were 45.87%, 3.49% and 2.49%. The apoptosis rates in the experimental group and the blank group were lower than that in the control group (all P<0.05). Transwell experimental results show that the number of cells passing through the cell membrane was (206±22), the blank group and the control group were (329±12) and (347±21). Significant difference was found in the number of cells between the experimental group and the blank group, the control group (all P<0.05). Conclusion After silencing S100A4 gene expression, the apoptosis of CNE2 cells was increased, and cell invasion ability was decreased.

Key words: nasopharyngeal neoplasms; nasopharyngeal carcinoma; calcium ion associated protein; S100A4 protein; apoptosis; cell invasion

鼻咽癌早期診斷率低，且易發(fā)生擴(kuò)散及遠(yuǎn)處轉(zhuǎn)移[1]。在腫瘤的發(fā)生發(fā)展及轉(zhuǎn)移過(guò)程中，存在某些基因及相關(guān)活化分子的激活[2]。鈣離子相關(guān)蛋白(S100A4蛋白)是一種鈣離子結(jié)合蛋白，能結(jié)合多種細(xì)胞內(nèi)靶蛋白并調(diào)節(jié)其功能，參與細(xì)胞增殖、分化、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞黏附、細(xì)胞外基質(zhì)重建及細(xì)胞運(yùn)動(dòng)等過(guò)程[3]，并與腫瘤的進(jìn)展有關(guān)。S100A4在鼻咽癌中的作用機(jī)制尚不明確，為此，2014年1月~2015年5月，我們觀察了沉默S100A4表達(dá)對(duì)鼻咽癌細(xì)胞CNE2凋亡及侵襲的影響，初步探討S100A4在鼻咽癌發(fā)生發(fā)展中的作用機(jī)制。

1材料與方法

1.1材料人鼻咽癌CNE2細(xì)胞株購(gòu)自廣西醫(yī)科大學(xué)耳鼻喉實(shí)驗(yàn)中心；Transwell培養(yǎng)板購(gòu)于美國(guó)Corning公司；Lipofectamine2000購(gòu)于美國(guó)Invitrogen公司；改良型1640培養(yǎng)基購(gòu)于Hyclone公司；胎牛血清購(gòu)自杭州四季青公司；胰酶、Tris、APS、SDS及TEMED購(gòu)于Sigma公司；Anecxin-V/PI凋亡試劑盒購(gòu)于美國(guó)Biovision公司；S100A4、GAPDH等相關(guān)抗體購(gòu)于Proteinch公司；S100A4 siRNA和空載體NC siRNA購(gòu)自維爾生物科技有限公司。

1.2CNE2細(xì)胞分組與S100A4 siRNA轉(zhuǎn)染將CNE2細(xì)胞培養(yǎng)于含12.5% FBS的1640培養(yǎng)基，置于37 ℃、5% CO2、飽和濕度培養(yǎng)箱中培養(yǎng)。細(xì)胞融合達(dá)80%左右用胰酶消化細(xì)胞，1∶2進(jìn)行傳代。待細(xì)胞密度為50%~70%時(shí)胰酶消化。準(zhǔn)備6孔板，各加入細(xì)胞懸液2 mL，繼續(xù)于培養(yǎng)箱中培養(yǎng)，轉(zhuǎn)染前更換為不含抗生素的培養(yǎng)基800 mL。準(zhǔn)備三支試管，標(biāo)注A管、B管、C管，分別配制三種溶液。A管溶液：將10 μL Lipofectamine2000轉(zhuǎn)染試劑移入200 μL無(wú)血清培養(yǎng)基中，輕晃混勻5 min；B管溶液：稀釋10 μL S100A4 siRNA于100 μL無(wú)血清培養(yǎng)基中，輕晃混勻5 min；C管溶液：稀釋10 μL空質(zhì)粒于100 μL無(wú)血清培養(yǎng)基中，輕晃混勻5 min。取A管溶液各100 μL分別加入B管、C管溶液中輕輕混勻。靜置20 min，將脂質(zhì)體復(fù)合物加入到對(duì)應(yīng)培養(yǎng)瓶中。將細(xì)胞隨機(jī)分為空白組(加等量無(wú)血清培養(yǎng)基，不做轉(zhuǎn)染)、對(duì)照組和實(shí)驗(yàn)組。6 h后，將Lipofectamine2000培養(yǎng)基更換為完全新鮮培養(yǎng)基，繼續(xù)培養(yǎng)48 h后取出，進(jìn)行后續(xù)實(shí)驗(yàn)。

1.3CNE2細(xì)胞中S100A4 mRNA檢測(cè)取三組細(xì)胞，用TRIzol試劑盒提取細(xì)胞總RNA，以總RNA為模板，逆轉(zhuǎn)錄成cDNA。S100A4 mRNA上游引物序列為5′-GCCCTGGATGTGATGGTGTC-3′，下游引物序列為5′-CCTCGTTGTCCCTGTTGCTGT-3′；內(nèi)參GAPDH mRNA上游引物序列為5′-CGGCAAATTCAACGGCACA-3′，下游引物序列為5′-GGTCTCGCTCCTGGAAGATGG-3′。進(jìn)行real-time PCR反應(yīng)。反應(yīng)條件：95 ℃ 10 min，95 ℃ 5 s，60 ℃ 10 s，72 ℃ 20 s，共40個(gè)循環(huán)。計(jì)算2-ΔCt，以此作為目的基因相對(duì)表達(dá)量。

1.4CNE2細(xì)胞中S100A4蛋白檢測(cè)準(zhǔn)備三組細(xì)胞，加入RIPA裂解液后反復(fù)吹打，提取蛋白，用BCA蛋白定量試劑盒定量蛋白，SDS聚丙烯酰胺凝膠電泳，轉(zhuǎn)接到PVDF膜，用5%脫脂奶粉進(jìn)行封閉，室溫放置1 h。加入一抗共孵育，4 ℃過(guò)夜，1×TBST漂洗3次。加入二抗，共孵育45~60 min。加入ECL化學(xué)發(fā)光液進(jìn)行孵育，在暗盒內(nèi)與X膠片曝光，顯影沖洗，采集圖像，采用Quantity One灰度分析軟件進(jìn)行分析、定量。

1.5凋亡CNE2細(xì)胞檢測(cè)用不含EDTA的胰酶消化各組細(xì)胞，PBS預(yù)冷洗滌，2 000 r/min離心5 min，棄上清液，重復(fù)2次，收集(1~5)×105個(gè)細(xì)胞，加Binding buffer 500 μL懸浮細(xì)胞，加入Annexin V-FITC 5 μL混勻后，再加Propidium Iodide 5 μL混勻。室溫下避光反應(yīng)5~15 min，1 h內(nèi)用流式細(xì)胞儀觀察細(xì)胞凋亡情況。

1.6CNE2細(xì)胞侵襲能力檢測(cè)將各組細(xì)胞用0.25%胰酶消化，用無(wú)血清培養(yǎng)基制成細(xì)胞懸液，調(diào)整細(xì)胞密度為1×106/mL，吸取100 μL加入侵襲小室內(nèi)，下室加入含10%FBS的DMEM，培養(yǎng)48 h。棄去小室內(nèi)培養(yǎng)液，用PBS洗2遍。用濕棉簽擦盡上室細(xì)胞，加入按1∶1混合的丙酮與甲醇溶液固定20 min，PBS洗2遍，用0.1%結(jié)晶紫染色20 min，清水洗3遍以上，顯微鏡下觀察，拍照，計(jì)數(shù)穿膜細(xì)胞。

1.7統(tǒng)計(jì)學(xué)方法采用細(xì)胞SPSS17.0統(tǒng)計(jì)軟件。計(jì)量資料以±s表示，多組間比較采用方差分析，兩組比較采用t檢驗(yàn)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2結(jié)果

2.1各組細(xì)胞S100A4 mRNA、蛋白表達(dá)比較實(shí)驗(yàn)組、空白組、對(duì)照組S100A4 mRNA相對(duì)表達(dá)量分別為0.600 4±0.05、1.000 0±0.00、0.894 1±0.09，S100A4 蛋白相對(duì)表達(dá)量分別為0.22±0.016、0.42±0.022、0.39±0.022。實(shí)驗(yàn)組S100A4 mRNA、蛋白表達(dá)量與空白組及對(duì)照組相比，P均<0.05。

2.2各組細(xì)胞凋亡情況比較實(shí)驗(yàn)組早期凋亡細(xì)胞占32.43%、晚期凋亡細(xì)胞占12.05%、死亡細(xì)胞占1.39%，凋亡率為45.87%；空白組細(xì)胞凋亡率為3.49%；對(duì)照組細(xì)胞凋亡率為2.49%。實(shí)驗(yàn)組與空白組及對(duì)照組相比，P均<0.05。

2.3各組細(xì)胞侵襲能力比較Transwell實(shí)驗(yàn)結(jié)果示實(shí)驗(yàn)組穿膜細(xì)胞數(shù)為(206±22)個(gè)，空白組和對(duì)照組分別為(329±12)、(347±21)個(gè)。實(shí)驗(yàn)組穿膜細(xì)胞數(shù)與空白組、對(duì)照組相比，P均<0.05。

3討論

S100基因家族主要分布在細(xì)胞質(zhì)及細(xì)胞核中，相對(duì)分子量在9~13 kD，編碼鈣離子結(jié)合蛋白[4]。S100A4是S100家族中的一員，由兩個(gè)同源二聚體結(jié)構(gòu)相捆綁形成，其中兩個(gè)亞單位呈反向平行，每個(gè)亞單位結(jié)構(gòu)的 EF雙螺旋手臂兩側(cè)的螺旋結(jié)構(gòu)區(qū)為疏水區(qū)，中間的環(huán)形結(jié)構(gòu)為鈣離子高度選擇性的親水區(qū)。當(dāng)與鈣離子結(jié)合后，S100A4蛋白構(gòu)象改變，暴露相關(guān)靶蛋白結(jié)合位點(diǎn)，遂與靶蛋白結(jié)合發(fā)揮相應(yīng)生物學(xué)作用[5]。研究表明，S100A4蛋白可通過(guò)鈣離子相關(guān)信號(hào)轉(zhuǎn)導(dǎo)通路參與細(xì)胞黏附、凋亡、基質(zhì)降解和血管生成，與多種腫瘤的發(fā)生發(fā)展有關(guān)[6，7]。在人類多種惡性腫瘤(如胃癌、直腸癌、乳腺癌、子宮內(nèi)膜癌、肺癌、膀胱癌等)組織中，S100A4蛋白表達(dá)增高，患者預(yù)后不理想[8]。因此，通過(guò)檢測(cè)S100A4蛋白有助于評(píng)估腫瘤侵襲、轉(zhuǎn)移等情況[9]。

有研究[10]表明，S100A4與P53結(jié)合后，可促使野生型P53丟失，P53正常結(jié)構(gòu)破壞、功能改變，導(dǎo)致細(xì)胞磷酸化作用發(fā)生異常，細(xì)胞的正常修復(fù)監(jiān)控功能喪失，發(fā)生惡變[11]，因此，沉默S100A4基因有可能抑制腫瘤細(xì)胞增殖。S100A4可激活MMP-2酶原成為有活性的MMP-2，還可通過(guò)激活NF-κB通路來(lái)誘導(dǎo)MMP-2合成，進(jìn)而使細(xì)胞外基質(zhì)發(fā)生降解，為腫瘤的侵襲與轉(zhuǎn)移提供條件[12，13]。還有研究表明，S100A4與鈣黏蛋白在腫瘤細(xì)胞黏附力方面分別起著正性、負(fù)性調(diào)節(jié)作用。當(dāng)S100A4表達(dá)上調(diào)時(shí)，鈣黏蛋白表達(dá)下降，腫瘤細(xì)胞間黏附作用減弱，易發(fā)生分化及轉(zhuǎn)移[14，15]，推測(cè)S100A4也可能通過(guò)影響MMP和鈣黏蛋白的分布從而促進(jìn)腫瘤細(xì)胞侵襲、轉(zhuǎn)移[16]。

本研究中，我們采用RNA干擾技術(shù)成功使CNE2細(xì)胞中S100A4 mRNA及蛋白表達(dá)下調(diào)，實(shí)驗(yàn)組細(xì)胞凋亡率高于空白組和對(duì)照組，細(xì)胞侵襲能力低于空白組及對(duì)照組。上述結(jié)果證實(shí)S100A4基因?qū)Ρ茄拾┘?xì)胞增殖和侵襲有促進(jìn)作用，而沉默S100A4基因表達(dá)后，鼻咽癌細(xì)胞凋亡增多，細(xì)胞侵襲能力下降，這為進(jìn)一步了解S100A4在鼻咽癌發(fā)生發(fā)展中的作用機(jī)制奠定了實(shí)驗(yàn)基礎(chǔ)。

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