共刺激分子B7-H3對(duì)食管癌細(xì)胞Eca-109生物學(xué)特性的影響

河北醫(yī)科大學(xué)第四醫(yī)院科研中心，河北 石家莊 050011

共刺激分子B7-H3對(duì)食管癌細(xì)胞Eca-109生物學(xué)特性的影響

曹娜娜 王玲 單保恩

河北醫(yī)科大學(xué)第四醫(yī)院科研中心，河北 石家莊 050011

背景與目的：食管腫瘤嚴(yán)重威脅著人類健康，認(rèn)識(shí)食管癌的發(fā)生、發(fā)展機(jī)制和尋找治療方法已經(jīng)成為遏制腫瘤的研究熱點(diǎn)。近年來(lái)，作為B7免疫球蛋白超家族的新成員，共刺激分子B7-H3在多種腫瘤中異常高表達(dá)，被認(rèn)為可能是一種新的腫瘤標(biāo)志物和潛在的治療靶點(diǎn)。本研究旨在檢測(cè)食管癌細(xì)胞株TE-1、TE-13、Eca-109細(xì)胞中B7-H3的表達(dá)，并通過(guò)靶向干擾B7-H3基因表達(dá)來(lái)研究B7-H3對(duì)食管癌細(xì)胞增殖、侵襲等生物學(xué)行為的影響。方法：運(yùn)用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcription polymerase chain reaction，RT-PCR)方法檢測(cè)B7-H3分子在食管癌細(xì)胞TE-1、TE-13、Eca-109中的表達(dá)。通過(guò)LipofectamineTM2000體外轉(zhuǎn)染B7-H3 siRNA、control siRNA至食管癌Eca-109細(xì)胞，采用RT-PCR和蛋白質(zhì)印跡法(Western blot)檢測(cè)Eca-109細(xì)胞中B7-H3 mRNA和蛋白的表達(dá)。采用MTT實(shí)驗(yàn)、細(xì)胞劃痕實(shí)驗(yàn)、Transwell侵襲小室實(shí)驗(yàn)檢測(cè)B7-H3 siRNA對(duì)Eca-109細(xì)胞增殖能力、平面遷移能力及體外侵襲能力的影響。結(jié)果：共刺激分子B7-H3在食管癌細(xì)胞TE-1(0.382±0.008)、TE-13(0.399±0.008)、Eca-109(0.428±0.012)中的表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。轉(zhuǎn)染B7-H3 siRNA后Eca-109細(xì)胞B7-H3 mRNA和蛋白表達(dá)水平顯著低于未轉(zhuǎn)染組(0.128 5±0.000 2 vs 0.540 3±0.001 3，0.421 4±0.004 8 vs 0.492 1±0.014 8，P均＜0.05)以及空載體轉(zhuǎn)染組(0. 128 5±0.000 2 vs 0.532 4±0.000 7，0.421 4±0.004 8 vs 0.500 6±0.012 9，P均＜0.05)。與對(duì)照組細(xì)胞相比，轉(zhuǎn)染B7-H3 siRNA后Eca-109細(xì)胞的平面遷移能力和侵襲力明顯下降(P＜0.05)，然而其增殖能力差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。結(jié)論：食管癌細(xì)胞TE-1、TE-13、Eca-109均組成性表達(dá)B7-H3分子。沉默B7-H3基因表達(dá)能明顯抑制Eca-109細(xì)胞的體外遷移、侵襲能力，提示B7-H3基因可能參與調(diào)節(jié)食管癌的侵襲轉(zhuǎn)移能力，為免疫治療提供潛在的治療靶點(diǎn)。

共刺激分子；B7-H3；食管癌；Eca-109

食管癌是我國(guó)最為高發(fā)的惡性腫瘤之一，每年平均約有20萬(wàn)人死于食管癌［1］。盡管近年來(lái)食管癌在診斷、治療等方面取得了巨大成就，但食管癌的預(yù)后仍然難盡人意。因此，了解食管癌發(fā)生、發(fā)展的分子機(jī)制對(duì)疾病的治療非常重要。

協(xié)同刺激分子B7-H3是新近發(fā)現(xiàn)的B7家族重要成員［2］，其在樹(shù)突狀細(xì)胞以及肝、肺、前列腺和相似腫瘤細(xì)胞系中均有表達(dá)［3-6］。然而，B7-H3的病理生理學(xué)作用機(jī)制尚未明確。有研究證明，B7-H3在大多情況下發(fā)揮抑制T細(xì)胞活性作用［7］。Chen等［8］發(fā)現(xiàn)B7-H3可能是一種腫瘤相關(guān)抗原，不僅可以調(diào)節(jié)重要細(xì)胞免疫應(yīng)答，還可以參與調(diào)節(jié)細(xì)胞生長(zhǎng)、凋亡、黏附及轉(zhuǎn)移等生物學(xué)特性［9-10］，提示其在腫瘤進(jìn)展中發(fā)揮重要作用［11］。本研究旨在檢測(cè)協(xié)同刺激分子B7-H3在食管癌細(xì)胞中的表達(dá)并探討其對(duì)食管癌細(xì)胞生物學(xué)特性的影響。

1 材料和方法

1.1 主要試劑

TRIzol、B7-H3引物、GAPDH引物、LipofectamineTM2000購(gòu)自美國(guó)Invitrogen公司，逆轉(zhuǎn)錄試劑盒Go Taq?GreenMaster Mix購(gòu)自Thermo公司，兔抗人B7-H3單克隆抗體和兔抗人GAPDH單克隆抗體購(gòu)自Epitomics公司，熒光標(biāo)記的羊抗兔的二抗購(gòu)自Rockland公司，MTT購(gòu)自北京索萊寶科技有限公司，B7-H3 shRNA、control shRNA購(gòu)自德國(guó)QIAGENE公司。Matrigel膠購(gòu)自美國(guó)BD Bioscience公司。Transwell侵襲小室購(gòu)自美國(guó)Costar公司。

1.2 細(xì)胞培養(yǎng)與基因轉(zhuǎn)染

TE-1、TE-13細(xì)胞由河北醫(yī)科大學(xué)第四醫(yī)院科研中心提供，Eca-109細(xì)胞購(gòu)自中國(guó)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。在含10%胎牛血清、青霉素(100 U/mL)、鏈霉素(100 μg/mL)的RPMI-1640培養(yǎng)基中，于37 ℃、CO2體積分?jǐn)?shù)為5%的飽和濕度條件下培養(yǎng)，細(xì)胞生長(zhǎng)呈70%～80%融合狀態(tài)時(shí)，以0.25%胰蛋白酶消化傳代，隔日換液，每3～4 d傳代1次，收集對(duì)數(shù)生長(zhǎng)期細(xì)胞進(jìn)行實(shí)驗(yàn)。利用LipofectamineTM2000轉(zhuǎn)染試劑，按照試劑盒所提供的說(shuō)明將B7-H3 siRNA、control siRNA基因轉(zhuǎn)染Eca-109細(xì)胞，實(shí)驗(yàn)設(shè)未轉(zhuǎn)染組、空載體轉(zhuǎn)染組(2.5 μg)、單獨(dú)轉(zhuǎn)染B7-H3 siRNA組(2.5 μg)。

1.3 逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcription polymerase chain reaction，RT-PCR)檢測(cè)食管癌細(xì)胞中B7-H3 mRNA的表達(dá)

收集TE-1、TE-13、Eca-109細(xì)胞以及轉(zhuǎn)染48 h后的Eca-109細(xì)胞。按照TRIzol試劑說(shuō)明書(shū)逐步加入氯仿、異丙醇、乙醇等提取總RNA，并測(cè)定RNA濃度、純度和完整性。取5 g總RNA按Revert Aid First Strand cDNA Synthesis Kit逆轉(zhuǎn)錄試劑盒(Fermantas公司)說(shuō)明書(shū)合成cDNA，用Go Taq? Green Master Mix進(jìn)行PCR擴(kuò)增。引物及反應(yīng)條件見(jiàn)表1。PCR產(chǎn)物經(jīng)1.5%瓊脂糖凝膠電泳分離后，紫外透射反射儀下觀察結(jié)果，并在凝膠成像儀上照相，掃描所得灰度值進(jìn)行統(tǒng)計(jì)學(xué)分析。

表1 RT-PCR的引物序列Tab. 1 Primer sequences for RT-PCR

1.4 蛋白質(zhì)印跡法(Western blot)檢測(cè)Eca-109細(xì)胞中B7-H3蛋白的表達(dá)

轉(zhuǎn)染48 h后，收集細(xì)胞，按RAPI∶PMSF=100∶1加入裂解混合液，冰上靜置20 min，在10 000×g條件下離心10 min，收集上清液并通過(guò)10%和15% SDS-PAGE定量蛋白，分離、轉(zhuǎn)膜、室溫封閉，兔抗人GAPDH單抗(1∶1 000)和兔抗人B7-H3單抗(1∶1 000) 4 ℃溫育過(guò)夜，TBST液洗膜3次，加入1∶10 000 TBST液稀釋的熒光素標(biāo)記抗兔二抗，37 ℃溫育1 h，TBST液洗膜3次，用雙色紅外熒光掃描系統(tǒng)成像并分析。

1.5 MTT檢測(cè)

Eca-109細(xì)胞轉(zhuǎn)染24 h后，將細(xì)胞以1.0×104個(gè)/孔接種在96孔板中，每組6個(gè)復(fù)孔培養(yǎng)過(guò)夜，細(xì)胞貼壁后0、24、48及72 h，每孔加入MTT溶液(5 mg/mL)20 μL繼續(xù)培養(yǎng)4 h，終止培養(yǎng)，小心吸棄孔內(nèi)培養(yǎng)上清液，每空加入150 μL二甲基亞砜(DMSO)，振蕩15 min，使結(jié)晶充分溶解，在酶聯(lián)免疫檢測(cè)儀上(490 nm)測(cè)定各孔光密度(A值)。取相同生長(zhǎng)天數(shù)及轉(zhuǎn)染相同質(zhì)粒A值的均值作為該組細(xì)胞某天的A值。

1.6 細(xì)胞劃痕實(shí)驗(yàn)

收集對(duì)數(shù)生長(zhǎng)期的Eca-109細(xì)胞，以2×105個(gè)/mL接種于24孔培養(yǎng)板中。待細(xì)胞長(zhǎng)至80%融合時(shí)，更換無(wú)血清的RPMI-1640，培養(yǎng)細(xì)胞24 h，用來(lái)饑餓細(xì)胞。待細(xì)胞長(zhǎng)到完全融合時(shí)，用10 μL加樣槍頭在每孔單層細(xì)胞上劃痕，造成培養(yǎng)細(xì)胞劃痕模型。劃痕后用PBS沖洗2次。沿劃痕邊緣等距離間隔做上標(biāo)記，以便檢測(cè)。實(shí)驗(yàn)分組同前，繼續(xù)培養(yǎng)24 h，定時(shí)用倒置顯微鏡觀察劃痕愈合程度并拍照。計(jì)算劃痕中間的距離，并與0 h的劃痕距離相比計(jì)算劃痕間距縮小的百分率，以此反映細(xì)胞的平面遷移能力。以上實(shí)驗(yàn)至少重復(fù)3次。

1.7 Transwell侵襲小室實(shí)驗(yàn)

實(shí)驗(yàn)采用帶有8 μm微孔聚碳酸酯膜的Transwell小室，將Matrigel (50 mg/L)膠和無(wú)血清RPMI-1640培養(yǎng)基以1∶3比例進(jìn)行稀釋。在小室上室鋪25 μL稀釋好的Matrigel膠，37 ℃無(wú)菌保持過(guò)夜，確保Matrigel膠充分聚合。收集對(duì)數(shù)生長(zhǎng)期的Eca-109細(xì)胞，用RPMI-1640培養(yǎng)基調(diào)整細(xì)胞濃度為2×105個(gè)/mL，每孔加入200 μL細(xì)胞懸液于上室，用RPMI-1640培養(yǎng)基補(bǔ)充至1 mL。下室加入預(yù)先培養(yǎng)Eca-109細(xì)胞24 h后的上清液，每孔加600 μL。實(shí)驗(yàn)分組同前。溫育約24 h后，取出上室。用棉簽頭擦掉Matrige膠及膠上細(xì)胞，PBS液洗3次。將小室置于4%多聚甲醛中固定15 min，風(fēng)干，然后用Gimesa染液染15～20 min，風(fēng)干。將聚碳酸酯膜用小剪刀剪下，放在載玻片上用樹(shù)膠封片，在顯微鏡下觀察結(jié)果，計(jì)數(shù)高倍鏡下至少5個(gè)視野內(nèi)的細(xì)胞數(shù)，取其平均值作為該組穿過(guò)Matrigel基質(zhì)的細(xì)胞數(shù)。

1.8 統(tǒng)計(jì)學(xué)處理

用SPSS 13.0統(tǒng)計(jì)學(xué)軟件對(duì)所有數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)處理，實(shí)驗(yàn)結(jié)果用表示，多組間均數(shù)比較采用單因素方差分析，P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 TE-1、TE-13和Eca-109細(xì)胞中B7-H3 mRNA的表達(dá)情況

RT-PCR檢測(cè)結(jié)果顯示，食管癌細(xì)胞TE-1(0.382±0.008)、TE-13(0.399±0.008)、Eca-109(0.428±0.012)均組成性表達(dá)B7-H3 mRNA (圖1)。B7-H3 mRNA在食管癌細(xì)胞Eca-109中的表達(dá)水平略高于其在TE-1、TE-13細(xì)胞中的表達(dá)，但差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。

圖1 TE-13、TE-1和Eca-109細(xì)胞中B7-H3的表達(dá)Fig. 1 B7-H3 expression in TE-13, TE-1 and Eca-109 cell lines

2.2 B7-H3 siRNA轉(zhuǎn)染Eca-109細(xì)胞后B7-H3 mRNA的表達(dá)情況

RT-PCR檢測(cè)結(jié)果顯示，B7-H3 mRNA在B7-H3 siRNA轉(zhuǎn)染組、空轉(zhuǎn)染組和未轉(zhuǎn)染組中的陽(yáng)性表達(dá)灰度值分別為0.128 5±0.000 2、 0.532 4±0.000 7和0.540 3±0.001 3。

統(tǒng)計(jì)分析結(jié)果發(fā)現(xiàn)，轉(zhuǎn)染B7-H3 siRNA后，B7-H3 mRNA表達(dá)水平明顯低于空轉(zhuǎn)染及未轉(zhuǎn)染組(P＜0.01)，說(shuō)明在轉(zhuǎn)錄水平上，B7-H3 siRNA轉(zhuǎn)染能下調(diào)B7-H3的轉(zhuǎn)錄活性(圖2、表2)。

圖2 B7-H3 siRNA轉(zhuǎn)染Eca-109細(xì)胞后B7-H3 mRNA的表達(dá)情況Fig. 2 B7-H3 mRNA expression in Eca-109 cells after B7-H3 siRNA transfection

表2 Eca-109細(xì)胞轉(zhuǎn)染后B7-H3 mRNA和蛋白表達(dá)水平Tab. 2 Expressions of B7-H3 mRNA and protein in Eca-109 cells with different transfection plasmids

2.3 B7-H3 siRNA轉(zhuǎn)染Eca-109細(xì)胞后B7-H3蛋白的表達(dá)情況

Western blot檢測(cè)結(jié)果顯示，B7-H3 siRNA轉(zhuǎn)染Eca-109細(xì)胞后，B7-H3蛋白的表達(dá)水平明顯低于空轉(zhuǎn)染組(0.421 4±0.004 8 vs 0.500 6± 0.012 9，P＜0.05)及未轉(zhuǎn)染組(0.421 4±0.004 8 vs 0.492 1±0.014 8，P＜0.05)，說(shuō)明在蛋白表達(dá)水平上，B7-H3 siRNA轉(zhuǎn)染下調(diào)了B7-H3的轉(zhuǎn)錄活性(圖3，表2)。

圖3 B7-H3 siRNA轉(zhuǎn)染Eca-109細(xì)胞后B7-H3蛋白的表達(dá)情況Fig. 3 B7-H3 protein expression in Eca-109 cells after B7-H3 siRNA transfection

2.4 B7-H3 siRNA轉(zhuǎn)染對(duì)Eca-109細(xì)胞增殖的影響

MTT分析結(jié)果顯示，轉(zhuǎn)染B7-H3 siRNA組、空載體組及未轉(zhuǎn)染細(xì)胞接種于96孔板24、48和72 h后，轉(zhuǎn)染B7-H3 siRNA的Eca-109細(xì)胞與空轉(zhuǎn)染及未轉(zhuǎn)染細(xì)胞相比，增殖率差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)，說(shuō)明B7-H3 siRNA轉(zhuǎn)染不能抑制Eca-109細(xì)胞增殖(圖4)。

圖4 B7-H3 siRNA轉(zhuǎn)染對(duì)Eca-109細(xì)胞增殖的影響Fig. 4 The effect of B7-H3 siRNA interference on Eca-109 cell proliferation

2.5 B7-H3 siRNA轉(zhuǎn)染對(duì)Eca-109細(xì)胞遷移能力的影響

劃痕實(shí)驗(yàn)結(jié)果顯示，未轉(zhuǎn)染組及空轉(zhuǎn)染組Eca-109細(xì)胞24 h后向劃痕邊緣爬行的速度快，劃痕的寬度明顯變窄，而轉(zhuǎn)染B7-H3 siRNA后Eca-109細(xì)胞向劃痕邊緣爬行的速度明顯減慢，劃痕缺損處修復(fù)緩慢，劃痕間距明顯縮短，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05，圖5)。提示轉(zhuǎn)染B7-H3 siRNA后，Eca-109細(xì)胞的平面運(yùn)動(dòng)能力明顯下降。

2.6 B7-H3 siRNA轉(zhuǎn)染對(duì)Eca-109細(xì)胞侵襲能力的影響

Transwell侵襲小室實(shí)驗(yàn)結(jié)果顯示，轉(zhuǎn)染B7-H3 siRNA后，通過(guò)人工基底膜的Eca-109細(xì)胞數(shù)明顯降低。與未轉(zhuǎn)染及空轉(zhuǎn)染組相比，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05，圖6)?？梢?jiàn)轉(zhuǎn)染B7-H3 siRNA后，Eca-109細(xì)胞的體外侵襲能力明顯減弱。

圖5 B7-H3 siRNA轉(zhuǎn)染對(duì)Eca-109細(xì)胞遷移能力的影響Fig. 5 The effect of B7-H3 siRNA interference on Eca-109 cell migration detected by wound scrape assay

圖6 B7-H3 siRNA轉(zhuǎn)染對(duì)Eca-109細(xì)胞體外侵襲能力的影響Fig. 6 The effect of B7-H3 siRNA interference on Eca-109 cell invasion ability detected by transwell invasion assay.

3 討 論

有研究證明B7-H3在腫瘤進(jìn)展和轉(zhuǎn)移中發(fā)揮重要作用。Wu等［12］在胃癌研究中證實(shí)，B7-H3表達(dá)與腫瘤浸潤(rùn)深度及生存時(shí)間相關(guān)。Sun等［13］發(fā)現(xiàn)，B7-H3在小細(xì)胞肺癌中高表達(dá)與淋巴結(jié)轉(zhuǎn)移風(fēng)險(xiǎn)的增加明顯相關(guān)。據(jù)報(bào)道，B7-H3過(guò)表達(dá)與腫瘤侵襲性和臨床預(yù)后有關(guān)，提示B7-H3在腫瘤進(jìn)展中發(fā)揮重要作用。

腫瘤發(fā)生是一個(gè)多步驟的過(guò)程，在這個(gè)過(guò)程中癌細(xì)胞增殖失控，從一個(gè)局限的初級(jí)的腫瘤團(tuán)塊擴(kuò)散侵襲附屬器官，再到遠(yuǎn)處器官轉(zhuǎn)移。腫瘤的侵襲和轉(zhuǎn)移主要包括細(xì)胞與基底膜的黏附、細(xì)胞外基質(zhì)的降解和細(xì)胞的遷移［14-15］。細(xì)胞無(wú)限制的生長(zhǎng)是癌細(xì)胞表型的一項(xiàng)重要改變［16］。本實(shí)驗(yàn)中，我們發(fā)現(xiàn)食管癌細(xì)胞TE-1、TE-13及Eca-109均組成性表達(dá)B7-H3蛋白。因此，我們通過(guò)干擾B7-H3基因的表達(dá)來(lái)研究其對(duì)食管癌細(xì)胞生長(zhǎng)特性的影響。轉(zhuǎn)染B7-H3 siRNA后Eca-109細(xì)胞的增殖能力與空轉(zhuǎn)染、未轉(zhuǎn)染細(xì)胞相比，有所下降，但差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。說(shuō)明B7-H3對(duì)食管癌細(xì)胞的增殖能力并無(wú)明顯影響。因此，我們用細(xì)胞的遷移、侵襲實(shí)驗(yàn)來(lái)驗(yàn)證基因敲除B7-H3是否夠能夠影響腫瘤細(xì)胞的散播轉(zhuǎn)移能力。

腫瘤細(xì)胞與原發(fā)部位分離，侵襲附屬器官及周邊組織時(shí)，需要一定的運(yùn)動(dòng)能力。細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示，轉(zhuǎn)染B7-H3 siRNA后，Eca-109細(xì)胞的平面爬行能力減弱，劃痕愈合變慢，提示轉(zhuǎn)染B7-H3 siRNA對(duì)細(xì)胞遷移能力有明顯抑制作用(P＜0.05)。腫瘤細(xì)胞受趨化因素趨化并穿過(guò)基底膜的能力是侵襲和轉(zhuǎn)移的關(guān)鍵因素之一。本研究采用正常培養(yǎng)食管癌細(xì)胞24 h的培養(yǎng)上清液作為趨化因素、Matrigel作為人工基底膜，通過(guò)Transwell侵襲小室觀察腫瘤細(xì)胞侵襲人工基底膜情況。結(jié)果表明，與空轉(zhuǎn)染、未轉(zhuǎn)染組相比，轉(zhuǎn)染B7-H3 siRNA組穿過(guò)基底膜的細(xì)胞數(shù)值顯著減少，提示B7-H3 siRNA轉(zhuǎn)染抑制B7-H3基因表達(dá)后，Eca-109細(xì)胞分解Matrigel膠的能力減弱，細(xì)胞穿過(guò)Transwell小室下表面的數(shù)目減少，Eca-109細(xì)胞的體外侵襲能力顯著降低。Tekle等［17］和Zhao等［18］也曾報(bào)道，干擾B7-H3基因能降低黑色素瘤、腎透明細(xì)胞癌等多種腫瘤細(xì)胞的遷移和侵襲能力。在Tekle等［17］的小鼠黑色素瘤模型中，沉默MDAMB-435細(xì)胞中B7-H3基因的表達(dá)能降低腫瘤轉(zhuǎn)移能力，顯著延長(zhǎng)小鼠的中位生存期。此外，在B7-H3基因沉默的小鼠體內(nèi)，基質(zhì)金屬蛋白酶2(matrix metalloproteinase，MMP-2)及IL-8的水平是降低的，而金屬蛋白酶抑制劑1(tissue inhibitor of metalloproteinase，TIMP-1)和2的水平是增加的。MMP-2與腫瘤的浸潤(rùn)及轉(zhuǎn)移密切相關(guān)，B7-H3可能通過(guò)抑制TIMP-1和TIMP-2的產(chǎn)生上調(diào)MMP-2的水平，促進(jìn)腫瘤侵襲與轉(zhuǎn)移的發(fā)生，提示B7-H3在調(diào)節(jié)腫瘤轉(zhuǎn)移過(guò)程中發(fā)揮重要作用，可能是抗轉(zhuǎn)移治療的潛在治療靶點(diǎn)。

綜上所述，靶向干擾B7-H3基因的表達(dá)能明顯抑制Eca-109細(xì)胞的遷移、侵襲能力，這提示B7-H3在腫瘤轉(zhuǎn)移中有重要作用，預(yù)示著高度侵襲能力和臨床不良預(yù)后。然而，B7-H3在體內(nèi)外發(fā)揮作用的分子機(jī)制仍需要進(jìn)一步研究。

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Effect of costimulatory molecule B7-H3 on the biological characteristics of esophageal cancer Eca-109 cell line

CAO Na-na, WANG Ling, SHAN Bao-en (Research Center, Fourth Hospital of Hebei Medical

University, Shijiazhuang Hebei 050011, China)

SHAN Bao-en E-mail: shanbaoen_1962@163.com

Background and purpose: Esophageal cancer is a serious disease threatening human health, and it is very difficult to understand the development mechanism and find the therapeutic methods for esophageal cancer. In recent years, B7-H3, as a new member of B7 immunoregulatory superfamily, overexpressed in multiple tumor types, is considered to be a new tumor marker and potential therapeutic target. This study aimed to detect the expression of B7-H3 in esophageal cancer cell lines TE-1, TE-13, Eca-109 and exploring the effect of B7-H3 siRNA on cell proliferation, migration and invasion in vitro in human esophageal cancer Eca-109 cell line. Methods: The expression of B7-H3 in esophageal cancer cell lines TE-1, TE-13 and Eca-109 were detected by reverse transcription polymerase chain reaction (RT-PCR). B7-H3 siRNA and control siRNA were transfected in vitro into human esophageal cancer Eca-109 cells using LipofectamineTM2000. The expressions of B7-H3 mRNA and protein in Eca-109 cells were analyzed by RT-PCR and Western blot. The proliferation, migration and invasion abilities of Eca-109 cells were measured by MTT assay, wound scrape assay and transwell invasion assay in vitro,respectively. Results: All tested cultured esophageal cancer cell lines constitutively expressed B7-H3 mRNA under normal conditions (TE-1 0.382±0.008, TE-13 0.399±0.008, Eca-109 0.428±0.012). After transfection, the expression of B7-H3 mRNA levels decreased in B7-H3 siRNA transfected group, compared with control siRNA transfected group (0.128 5±0.000 2 vs 0.532 4±0.000 7, P＜0.01) and untransfected group (0.128 5±0.000 2 vs 0.540 3±0.001 3, P＜0.01), while its protein expression levels were also signi ficantly lower than the control transfection group (0.421 4±0.004 8 vs 0.500 6±0.012 9, P＜0.05) and untransfected group (0.421 4±0.004 8 vs 0.492 1±0.014 8, P＜0.05). Compared with control transfected and untransfected cells, Eca-109 cell migration and invasion abilities decreased significantly (P＜0.05) by siRNA interference, but no significant difference was observed between their proliferative capacity (P＞0.05). Conclusion: All tested esophageal cancer cell lines constitutively express B7-H3 mRNA. B7-H3 siRNA interference inhibits Eca-109 cell migration and invasion abilities. B7-H3 may have a critical role in regulating Eca-109 cell progression.

Costimulatory molecule; B7-H3; Esophageal cancer; Eca-109

10.3969/j.issn.1007-3969.2014.08.001

R735.1

A

1007-3639(2014)08-0561-07

2014-04-24

2014-07-15）

國(guó)家自然資金面上項(xiàng)目(No：81173611)；河北省重大醫(yī)學(xué)科研課題(No：zd2013045)。

單保恩 E-mail:shanbaoen_1962@163.com