人巨細(xì)胞病毒通過 STAT3上調(diào)膠質(zhì)瘤中endocan表達(dá)

王士杰，范東瀛，羅喬荔，邢燕，王一松，安靜

首都醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院微生物學(xué)教研室，北京 100069

·論著·

人巨細(xì)胞病毒通過 STAT3上調(diào)膠質(zhì)瘤中endocan表達(dá)

王士杰，范東瀛，羅喬荔，邢燕，王一松，安靜

首都醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院微生物學(xué)教研室，北京 100069

本研究旨在探討人巨細(xì)胞病毒(human cytomegalovirus，HCMV)和內(nèi)皮細(xì)胞特異性分子1(endocan)在膠質(zhì)瘤發(fā)生發(fā)展中的作用及機(jī)制。利用HCMV感染人腦膠質(zhì)瘤U87細(xì)胞，檢測(cè)感染后第1、2、4天信號(hào)轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄激活蛋白 3(signal transducer and activator of transcription 3，STAT3)和endocan的表達(dá)變化，并分析HCMV、STAT3和endocan三者之間的可能關(guān)聯(lián)。結(jié)果顯示，HCMV感染的U87細(xì)胞中pSTAT3和endocan表達(dá)上調(diào)，并隨感染時(shí)間延長(zhǎng)而逐漸升高；用 RNAi干擾STAT3基因表達(dá)，endocan表達(dá)量下降；利用抗病毒藥物更昔洛韋抑制 HCMV 復(fù)制后，pSTAT3表達(dá)量下降，endocan表達(dá)量隨之下降。進(jìn)一步利用免疫組織化學(xué)法檢測(cè)79例腦膠質(zhì)瘤患者和8例對(duì)照者腦組織中pSTAT3表達(dá)情況，發(fā)現(xiàn)pSTAT3在膠質(zhì)瘤組織中表達(dá)上調(diào)；與低級(jí)別膠質(zhì)瘤相比，高級(jí)別膠質(zhì)瘤中pSTAT3染色強(qiáng)度較高。結(jié)果提示，HCMV感染可能通過STAT3信號(hào)通路上調(diào)endocan表達(dá)，從而參與膠質(zhì)瘤的進(jìn)展。

膠質(zhì)瘤；人巨細(xì)胞病毒；內(nèi)皮細(xì)胞特異性分子1；信號(hào)轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄激活蛋白3

膠質(zhì)瘤是發(fā)病率最高的顱內(nèi)腫瘤，具有高發(fā)病率、高復(fù)發(fā)率、高死亡率及低治愈率的特點(diǎn)。膠質(zhì)瘤的病因未明，可能與遺傳、環(huán)境及電離輻射等多種因素有關(guān)。近年來，病毒感染與腫瘤發(fā)生的關(guān)系成為研究熱點(diǎn)。2002年，Cobbs 等首次報(bào)道在不同級(jí)別的膠質(zhì)瘤組織中人巨細(xì)胞病毒(human cytomegalovirus，HCMV)IE1-72抗原和基因檢出率高達(dá)100%[1]。隨后一系列研究表明，HCMV基因序列和表達(dá)產(chǎn)物存在于大多數(shù)惡性膠質(zhì)瘤中，但缺少HCMV參與膠質(zhì)瘤進(jìn)展的直接證據(jù)[2]。

HCMV屬皰疹病毒科β亞科，是人皰疹病毒家族中基因組最大的病毒。HCMV在人群中的感染率高達(dá)60%～90%，且一旦感染終身攜帶。已有研究在多種腫瘤組織中檢出HCMV相關(guān)組分；同時(shí)發(fā)現(xiàn)HCMV可通過與一些關(guān)鍵信號(hào)通路相互作用而參與調(diào)節(jié)膠質(zhì)母細(xì)胞瘤的惡性表型[3-5]。已知信號(hào)轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄激活蛋白(signal transducer and activator of transcription， STAT)家族是一組可被多種細(xì)胞因子激活的轉(zhuǎn)錄因子。STAT3在人體正常組織及細(xì)胞中有少許表達(dá)，是胚胎發(fā)育、分化，特別是神經(jīng)干細(xì)胞和星形膠質(zhì)細(xì)胞發(fā)育的重要調(diào)節(jié)因子[6]。然而，在腫瘤組織及細(xì)胞中STAT3異常高表達(dá)[6]，其組成性激活(constitutive activation)參與腫瘤細(xì)胞的惡性侵襲和轉(zhuǎn)移[7]。HCMV是否通過激活STAT3信號(hào)通路參與膠質(zhì)瘤的發(fā)生發(fā)展，尚待進(jìn)一步研究。

豐富的新生血管是惡性膠質(zhì)瘤生長(zhǎng)、侵襲和轉(zhuǎn)移的重要病理學(xué)基礎(chǔ)之一，這一過程受血管生成因子調(diào)控。已知人內(nèi)皮細(xì)胞特異性分子1(endocan)是一個(gè)相對(duì)分子質(zhì)量為50 000的蛋白聚糖[8]。生理?xiàng)l件下，endocan由血管內(nèi)皮細(xì)胞表達(dá)，在健康人血液中僅可微量檢出。近年來研究表明，endocan與細(xì)胞黏附、炎癥反應(yīng)、腫瘤形成等過程有關(guān)[8-10]。炎性細(xì)胞因子如腫瘤壞死因子α(tumor necrosis factor α，TNF-α)、促血管生成因子〔如血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor，VEGF)、成纖維細(xì)胞生長(zhǎng)因子2(fibroblast growth factor 2，F(xiàn)GF-2)、肝細(xì)胞生長(zhǎng)因子/分裂因子(hepatocyte growth factor/scatter factor，HGF/SF)〕等可促進(jìn)endocan分泌[11]。另有研究表明，endocan在腫瘤患者的腫瘤組織和血液中表達(dá)增加[12-13]。在裸鼠荷瘤模型中，已證明endocan異位表達(dá)可促進(jìn)腫瘤生長(zhǎng)[14]。但endocan在膠質(zhì)瘤進(jìn)展中的作用尚未見報(bào)道。

本課題組前期研究發(fā)現(xiàn)，膠質(zhì)瘤組織中HCMV pp65和endocan表達(dá)明顯增加，且與膠質(zhì)瘤的病理級(jí)別密切關(guān)聯(lián)[15]，但HCMV是否與endocan相互作用及機(jī)制不明。為此，本研究通過體外實(shí)驗(yàn)探討HCMV感染對(duì)STAT3和endocan表達(dá)的影響及三者可能的調(diào)控關(guān)系，在此基礎(chǔ)上，用免疫組織化學(xué)方法檢測(cè)79例腦膠質(zhì)瘤標(biāo)本(腫瘤組織)及 8 例正常腦組織標(biāo)本中pSTAT3的表達(dá)水平及其與膠質(zhì)瘤病理級(jí)別的相關(guān)性。

1 材料與方法

1.1 臨床標(biāo)本

79 例膠質(zhì)瘤標(biāo)本來自首都醫(yī)科大學(xué)附屬北京天壇醫(yī)院神經(jīng)外科于2007年1月—2013年9月手術(shù)的患者，對(duì)照腦組織取自腦外傷手術(shù)患者。所有病例的蘇木精-伊紅(hematoxylin-eosin，HE)染色切片均由兩位病理科醫(yī)師讀片確認(rèn)為神經(jīng)膠質(zhì)瘤，并根據(jù)2007年世界衛(wèi)生組織(World Health Organization，WHO)中樞神經(jīng)系統(tǒng)腫瘤分類標(biāo)準(zhǔn)對(duì)所有標(biāo)本核對(duì)病理分級(jí)，其中低級(jí)別膠質(zhì)瘤(WHO Ⅰ～Ⅱ)36例、高級(jí)別膠質(zhì)瘤(WHO Ⅲ～Ⅳ) 43例。所選病例包括男性48例、女性31例；平均年齡43.8歲(8～78歲)?；颊咝g(shù)前未進(jìn)行任何特殊治療，也無其他系統(tǒng)腫瘤病史。本研究涉及個(gè)體均由本人或其監(jiān)護(hù)人簽署知情同意書，同時(shí)獲得首都醫(yī)科大學(xué)附屬北京天壇醫(yī)院倫理委員會(huì)的批準(zhǔn)。手術(shù)切除的組織標(biāo)本置于4%多聚甲醛中保存。

1.2 方法

1.2.1 主要試劑 抗-STAT3單克隆抗體(MAB1799)、抗pSTAT3多克隆抗體(AF4607)均購(gòu)自美國(guó)R&D 公司，抗endocan抗體(LIA-1001)購(gòu)自法國(guó)Lunginnov公司。STAT3-RNAi質(zhì)粒(9077)由上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司構(gòu)建，載體為GV248，嘌呤霉素抗性，可在脊椎動(dòng)物細(xì)胞內(nèi)瞬時(shí)或穩(wěn)定表達(dá)。

1.2.2 細(xì)胞株及病毒株 人胚肺成纖維細(xì)胞MRC-5購(gòu)于協(xié)和細(xì)胞資源中心，人膠質(zhì)瘤細(xì)胞系U87由首都醫(yī)科大學(xué)附屬北京天壇醫(yī)院王群教授惠贈(zèng)，HCMV AD169 株由中國(guó)醫(yī)科大學(xué)盛京醫(yī)院阮強(qiáng)教授惠贈(zèng)。

1.2.3 細(xì)胞感染實(shí)驗(yàn) 用HCMV AD169株感染U87細(xì)胞〔感染復(fù)數(shù)(multiplicity of infection，MOI)為3，于37 ℃ 孵育2 h〕，分別于感染后第1、2、4天收取細(xì)胞培養(yǎng)上清液，提取RNA，實(shí)時(shí)定量聚合酶鏈反應(yīng)(polymerase chain reaction，PCR)測(cè)定HCMV US17基因表達(dá)水平，以確定感染是否成功。同時(shí)，利用實(shí)時(shí)定量PCR檢測(cè)endocan和STAT3，并以β-actin為內(nèi)參(引物見表1)。同一時(shí)間點(diǎn)收取細(xì)胞內(nèi)蛋白，用蛋白免疫印跡法檢測(cè)endocan、STAT3及pSTAT3表達(dá)，并利用ImageJ2x軟件進(jìn)行半定量分析。

表1 PCR使用的引物序列

Tab.1 Primers used in PCR

GeneSequenceProductsize(bp)Human?actin?F5′?TGGCACCCAGCACAATGAA?3′188Human?actin?R5′?CTAAGTCATAGTCCGCCTAGAAGCA?3′STAT3?F5′?TGCGGAGAAGCATCGTGAGT?3′124STAT3?R5′?CCTCCAATGCAGGCAATCTGT?3′Endocan?F5′?CAGGCATGGATGGCATGAAG?3′150Endocan?R5′?CTGACTGGCAGTTGCAGGTCTC?3′US17?F5′?GCGTGCTTTTTAGCCTCTGCA?3′152US17?R5′?AAAGTTTGTGCCCCAACGGTA?3′

1.2.4 STAT3-siRNA細(xì)胞株建立 STAT3-RNAi質(zhì)粒由上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司構(gòu)建，包含針對(duì)STAT3 mRNA序列的shRNA編碼克隆，序列為5′-TGACCAACAATCCCAAGAA-3′。分別提取STAT3-RNAi質(zhì)粒和對(duì)照質(zhì)粒，脂質(zhì)體(LipofectamineTM2000)轉(zhuǎn)染U87細(xì)胞。用0.5 g/mL嘌呤霉素對(duì)細(xì)胞進(jìn)行篩選，獲得陽性克隆細(xì)胞株，分別命名為U87-STAT3-down和U87-STAT3-control。 用蛋白免疫印跡法及定量PCR檢測(cè)U87-STAT3-down 細(xì)胞株中STAT3的水平變化，并與U87-STAT3-control比較，以確定轉(zhuǎn)染是否成功。

1.2.5 更昔洛韋毒性實(shí)驗(yàn)與更昔洛韋抗病毒實(shí)驗(yàn) 用四甲基偶氮唑鹽(methylthiazolyl tetrazolium，MTT)法檢測(cè)更昔洛韋對(duì)U87細(xì)胞的毒性作用，結(jié)果顯示更昔洛韋在 800 μmol/L 以內(nèi)無明顯細(xì)胞毒性作用。同時(shí)檢測(cè)更昔洛韋對(duì)HCMV的抑制作用，發(fā)現(xiàn)200 μmol/L即可明顯抑制HCMV復(fù)制，因此選取200 μmol/L為本研究中更昔洛韋的工作濃度。

HCMV感染 2 h后加入更昔洛韋(200 μmol/L)維持 4 d。于感染后第1、2、4天收集細(xì)胞，提取 RNA，實(shí)時(shí)定量PCR檢測(cè) U87 細(xì)胞中endocan 和STAT3 mRNA表達(dá)水平；RIPA裂解法提取總蛋白，蛋白免疫印跡法檢測(cè)endocan、STAT3及pSTAT3蛋白表達(dá)水平。

1.2.6 免疫組織化學(xué)染色 石蠟切片厚度為6 μm，采用常規(guī)方法進(jìn)行脫蠟，隨后用0.01 mol/L枸櫞酸鈉緩沖液進(jìn)行抗原修復(fù)，常規(guī)一抗(anti-pSTAT3單克隆抗體)、二抗孵育及顯色。顯微鏡下觀察染色情況，每張切片隨機(jī)取5個(gè)高倍視野，每個(gè)視野計(jì)數(shù)100個(gè)細(xì)胞，其中細(xì)胞質(zhì)或細(xì)胞核染成黃色或棕色的細(xì)胞為陽性細(xì)胞。根據(jù)切片中陽性細(xì)胞的表達(dá)強(qiáng)度和陽性細(xì)胞數(shù)分別評(píng)分。染色強(qiáng)度評(píng)分如下：無色為0分、淺黃色為1分、黃或棕色為2分、深棕色為3分；陽性細(xì)胞數(shù)評(píng)分如下：<5%為0分、5%～25%為1分、26%～50%為2分、51%～75%為3分、>75%為4分，并將每份標(biāo)本的兩個(gè)分值相乘作為該膠質(zhì)瘤中蛋白表達(dá)的相對(duì)量化指標(biāo)，得分≤3分為陰性，3分<得分≤6分為陽性，得分>6分為強(qiáng)陽性。

1.3 統(tǒng)計(jì)學(xué)處理

采用SPSS 17.0統(tǒng)計(jì)軟件進(jìn)行處理。計(jì)量資料以mean±SD表示，采用單因素方差分析和t檢驗(yàn)；計(jì)數(shù)資料采用卡方檢驗(yàn)。

2 結(jié)果

2.1 HCMV感染對(duì)U87細(xì)胞中endocan和pSTAT3表達(dá)的影響

用實(shí)時(shí)定量PCR檢測(cè)HCMV感染U87細(xì)胞中HCMV US17 mRNA表達(dá)情況。結(jié)果顯示，病毒感染后第1天US17 mRNA表達(dá)，變化不明顯，第2、4天表達(dá)水平明顯增加，與第1天相比，分別上升160%和330%，表明HCMV成功感染U87細(xì)胞(圖1)。

用實(shí)時(shí)定量PCR檢測(cè)HCMV感染U87細(xì)胞中endocan mRNA表達(dá)水平。與對(duì)照細(xì)胞相比，HCMV 感染后第1 天即可見 endocan mRNA表達(dá)水平上升25%，感染后第 2 和 4 天 endocan mRNA表達(dá)水平顯著升高，分別達(dá)270%和400%，與對(duì)照細(xì)胞相比有顯著差異(P<0.05和P<0.01)(圖 2A)。未感染HCMV的U87細(xì)胞在培養(yǎng)后第1、2和4天endocan mRNA水平無明顯變化，與生理?xiàng)l件下endocan mRNA表達(dá)極低一致。用蛋白免疫印跡法檢測(cè)endocan蛋白表達(dá)變化，結(jié)果與 mRNA 水平變化一致，在HCMV 感染后第 1天變化不顯著，第2天達(dá)峰值(P<0.01)，第4天仍維持在較高水平(P<0.05)，分別為對(duì)照細(xì)胞的1.6倍和1.3倍(圖2B)。

用蛋白免疫印跡法檢測(cè)HCMV感染U87細(xì)胞中STAT3及pSTAT3的表達(dá)變化， 結(jié)果如圖3所示，STAT3表達(dá)變化趨勢(shì)與pSTAT3一致。對(duì)pSTAT3的分析顯示，HCMV 感染 U87 細(xì)胞后第1天pSTAT3表達(dá)水平即升高1.7倍，與對(duì)照細(xì)胞相比有顯著差異(P<0.01)，且明顯早于endocan表達(dá)上調(diào)的時(shí)間；感染后第2、4天pSTAT3表達(dá)水平仍維持在較高水平，與對(duì)照細(xì)胞相比分別上調(diào)1.5倍和1.3倍。

HCMV US17 mRNA expression level in U87 cells was analyzed by real-time PCR. US17 mRNA expression level gradually increased at 1, 2, and 4 d post-infection. The treatment with ganciclovir (GCV) markedly decreased US17 mRNA expression level. Values represent mean±SD from at least three independent experiments. dpi, days post-infection.

圖1 HCMV感染U87細(xì)胞后HCMV US17基因mRNA水平變化及更昔洛韋處理對(duì)其影響

Fig.1 The changes in HCMV US17 mRNA level in HCMV-infected U87 cells treated with or without ganciclovir

A. Endocan mRNA level in HCMV-infected U87 cells analyzed by real-time PCR. B. Endocan protein level in HCMV-infected U87 cells analyzed by Western blotting. Upper panel: Representative images of endocan expression in HCMV-infected U87 cells. β-actin was used as a loading control. Lower panel: A densitometric analysis of Western blotting results (n=3). Values represent mean±SD from at least three independent experiments.*P<0.05,**P<0.01versusmock group as determined by the one-way analysis of variance. dpi, days post-infection.

圖2 HCMV感染U87細(xì)胞后endocan mRNA和蛋白表達(dá)水平上調(diào)

Fig.2 Upregulation of endocan expression level in HCMV-infected U87 cells

A. STAT3 expression level. Upper panel: Representative images of STAT3 and β-actin expression in HCMV-infected U87 cells. Lower panel: A densitometric analysis of Western blotting results. B. pSTAT3 expression level. Upper panel: Representative images of pSTAT3 and STAT3 expression in HCMV-infected U87 cells. Lower panel: A densitometric analysis of Western blotting results. Values represent mean±SD from at least three independent experiments.*P<0.05,**P<0.01versusmock group as determined by the one-way analysis of variance. dpi, days post-infection.

圖3 HCMV感染U87細(xì)胞后STAT3和pSTAT3的表達(dá)水平

Fig.3 The changes in STAT3 and pSTAT3 expression levels in HCMV-infected U87 cells

2.2 HCMV感染對(duì)STAT3下調(diào)細(xì)胞株中pSTAT3和endocan表達(dá)的影響

用STAT3-RNAi質(zhì)粒和對(duì)照質(zhì)粒分別轉(zhuǎn)染U87細(xì)胞，經(jīng)嘌呤霉素篩選，定量PCR發(fā)現(xiàn)STAT3 mRNA 水平與對(duì)照細(xì)胞(U87-STAT3-control)相比降低17%(P<0.05)(圖 4A)。與此相一致，STAT3蛋白表達(dá)量降低60%，與對(duì)照細(xì)胞相比有顯著差異(P<0.01)(圖 4B)。表明STAT3下調(diào)的U87細(xì)胞株構(gòu)建成功，命名為 U87-STAT3-down。

用實(shí)時(shí)定量PCR檢測(cè)U87-STAT3-down細(xì)胞中endocan mRNA表達(dá)水平變化，結(jié)果如圖 4A所示。與U87-STAT3-control相比，在U87-STAT3-down細(xì)胞中STAT3表達(dá)下降的同時(shí)，endocan mRNA表達(dá)水平降低至43%(圖4A)，蛋白表達(dá)水平顯示一定的下降趨勢(shì)(圖4B)，提示STAT3可能調(diào)控endocan的表達(dá)。

進(jìn)一步用蛋白免疫印跡法檢測(cè)HCMV感染后第2天U87-STAT3-down和U87-STAT3-control細(xì)胞中endocan和STAT3蛋白表達(dá)水平，結(jié)果顯示兩種蛋白表達(dá)水平明顯低于感染對(duì)照組，分別為對(duì)照組的80% 和43%，與感染的STAT3-control細(xì)胞相比具有顯著差異(P<0.05)(圖5)。感染并未引起U87-STAT3-down細(xì)胞中STAT3和endocan蛋白上調(diào)，提示STAT3下調(diào)可能阻斷HCMV感染引起的endocan上調(diào)。

2.3 更昔洛韋對(duì)U87細(xì)胞中endocan和pSTAT3表達(dá)的影響

在HCMV感染后2 h，加入200 μmol/L更昔洛韋處理感染細(xì)胞，并維持至所觀察的時(shí)間點(diǎn)。結(jié)果顯示，處理后細(xì)胞培養(yǎng)上清液中US17 mRNA表達(dá)水平較HCMV感染組明顯下降，表明更昔洛韋能有效抑制 HCMV 復(fù)制(圖 1)。利用實(shí)時(shí)定量PCR檢測(cè)STAT3和endocan mRNA轉(zhuǎn)錄水平，結(jié)果顯示，在HCMV感染后第1、2、4天，更昔洛韋處理組STAT3和endocan mRNA表達(dá)水平均下調(diào)(P<0.05，P<0.01)(圖6A)。與此相一致，pSTAT3和endocan蛋白表達(dá)水平也呈現(xiàn)不同程度下降。更昔洛韋處理組endocan蛋白表達(dá)量在感染后第1天降低至HCMV感染組的85%，在感染第2、4天下降更顯著，分別降至40%和50%(P<0.01)；pSTAT3蛋白表達(dá)量明顯下降出現(xiàn)在HCMV感染后第1、2天(P<0.05)，第4天略有恢復(fù)，降至HCMV感染組的85%(圖6B)。

A: Expression levels of endocan and STAT3 mRNA in U87-STAT3-down cells analyzed by real-time PCR. RNAi-STAT3 downregulated endocan mRNA level. B: Expression levels of endocan and STAT3 proteins in U87-STAT3-down cells analyzed by Western blotting. Downregulated endocan and STAT3 were observed in U87-STAT3-down cells. Upper panel: Representative images of endocan and STAT3 expression in U87-STAT3-down cells. β-actin was used as a loading control. Lower panel: A densitometric analysis of Western blotting results (n=3). Values represent mean±SD from at least three independent experiments.*P<0.05,**P<0.01versusSTAT3-control group as determined by the one-way analysis of variance. dpi, days post-infection.

圖4 U87-STAT3-down細(xì)胞的鑒定及endocan和STAT3的表達(dá)變化

Fig.4 Identification of U87-STAT3-down cell line and changes in endocan and STAT3 expression levels

The expression levels of endocan and STAT3 in U87-STAT3-down cells at 2 d post-infection were analyzed by Western blotting. The expression levels of endocan and STAT3 were downregulated in U87-STAT3-down cells. Upper panel: Representative images of endocan and STAT3 expression in U87-STAT3-down cells. β-actin was used as a loading control. Lower panel: A densitometric analysis of Western blotting results (n=3). Values represent mean±SD from at least three independent experiments.*P<0.05,**P<0.01versusSTAT3-control group as determined by the one-way analysis of variance. dpi, days post-infection.

圖5 HCMV感染后第2天U87-STAT3-down細(xì)胞中endocan和STAT3的表達(dá)變化

Fig.5 The changes in endocan and STAT3 expression levels in U87-STAT3-down cells at 2 d post-infection

A: Endocan and STAT3 mRNA expression levels determined at 1, 2 and 4 dpi in HCMV-infected U87 cells treated with GCV (+) or DMSO as a control (-). B: Western blotting analysis of pSTAT3, STAT3 and endocan in HCMV-infected U87 cells treated with GCV (+) or DMSO as a control (-) at 1, 2 and 4 dpi. Upper panel: Representative images of pSTAT3, STAT3 and endocan expression in HCMV-infected U87 cells with or without GCV treatment. β-actin was used as a loading control. Lower panel: A densitometric analysis of Western blotting results. Values represent mean±SD from at least three independent experiments.*P<0.05,**P<0.01versuscontrol group as determined by the one-way analysis of variance. dpi, days post-infection.

圖6 更昔洛韋處理對(duì)HCMV感染的U87細(xì)胞中endocan 和 STAT3表達(dá)水平的影響

Fig.6 The changes in endocan and pSTAT3 expression levels in HCMV-infected U87 cell after treatment with ganciclovir

The expression level of pSTAT3 protein in gliomas was detected by IHC. Representative images were shown at low (upper panel) or high (lower panel) magnification. The positive staining of pSTAT3 was predominantly observed in the cytoplasm of tumor cells. The strong intensity of the staining was closely associated with high grade of glioma (grades III and IV). No expression of pSTAT3 was detected in the control brain tissues. Scale bar, 100 μm.

圖7 免疫組織化學(xué)法檢測(cè)膠質(zhì)瘤和對(duì)照腦組織中pSTAT3蛋白的表達(dá)

Fig.7 The expression level of pSTAT3 in gliomas and control brain tissues

2.4 pSTAT3蛋白在膠質(zhì)瘤腦組織中的表達(dá)

用免疫組織化學(xué)法對(duì)79例膠質(zhì)瘤石蠟標(biāo)本中的pSTAT3表達(dá)情況進(jìn)行檢測(cè)。結(jié)果顯示，對(duì)照腦組織中僅有微量pSTAT3表達(dá)，而膠質(zhì)瘤標(biāo)本中pSTAT3表達(dá)水平明顯增加，主要分布于腫瘤細(xì)胞的細(xì)胞核內(nèi)，細(xì)胞質(zhì)中可見少許pSTAT3陽性反應(yīng)。與低級(jí)別膠質(zhì)瘤相比，高級(jí)別膠質(zhì)瘤中pSTAT3染色強(qiáng)度較高(圖7)。

3 討論

惡性膠質(zhì)瘤是神經(jīng)系統(tǒng)腫瘤中發(fā)病率最高的顱內(nèi)腫瘤，預(yù)后不良。其發(fā)病原因不明，可能與多種因素有關(guān)，如遺傳、環(huán)境因素、病毒感染等。自2002年Cobbs首次報(bào)道HCMV在100%的膠質(zhì)瘤組織中被檢出后，HCMV在膠質(zhì)瘤發(fā)生中的作用和機(jī)制倍受關(guān)注。本課題組在前期研究中發(fā)現(xiàn)，膠質(zhì)瘤組織中HCMV pp65和endocan表達(dá)明顯增加，且與膠質(zhì)瘤的病理級(jí)別成正比[15]，支持HCMV參與膠質(zhì)瘤發(fā)生的觀點(diǎn)，且可能與endocan密切關(guān)聯(lián)，但分子機(jī)制尚不清楚。已知STAT3信號(hào)通路是調(diào)控細(xì)胞生長(zhǎng)、分化等多種功能的關(guān)鍵分子，其磷酸化后轉(zhuǎn)位入核，參與調(diào)控細(xì)胞存活、分化、增殖，血管生成和免疫功能的多種基因表達(dá)[16]，與多種細(xì)胞生物學(xué)時(shí)間密切相關(guān)。有趣的是，Cobbs等研究發(fā)現(xiàn)HCMV US28蛋白在神經(jīng)前細(xì)胞中可誘導(dǎo)STAT3活化，因而推測(cè)HCMV、STAT3、endocan三者之間可能存在某種關(guān)聯(lián)。為驗(yàn)證此推論，本研究利用HCMV感染膠質(zhì)瘤U87細(xì)胞，發(fā)現(xiàn)HCMV 感染可誘導(dǎo)U87細(xì)胞中endocan mRNA和蛋白表達(dá)水平上調(diào)；利用RNAi下調(diào)U87細(xì)胞中STAT3表達(dá)(U87-STAT3-down)后，endocan表達(dá)呈現(xiàn)明顯下降，因此利用RNAi下調(diào)STAT3表達(dá)，可阻斷HCMV感染引起的endocan水平上調(diào)。進(jìn)一步加入更昔洛韋抑制HCMV復(fù)制，U87細(xì)胞中endocan mRNA和蛋白表達(dá)水平下降，同時(shí)pSTAT3表達(dá)下調(diào)。以上結(jié)果提示，HCMV感染可能通過STAT3上調(diào)endocan表達(dá)參與膠質(zhì)瘤的進(jìn)展。進(jìn)一步利用膠質(zhì)瘤組織標(biāo)本檢測(cè)pSTAT3(活性STAT3)的表達(dá)情況，發(fā)現(xiàn)pSTAT3在膠質(zhì)瘤組織中高表達(dá)，且表達(dá)強(qiáng)度與腫瘤級(jí)別相關(guān)聯(lián)。

新生血管生成是腫瘤生長(zhǎng)與侵襲的關(guān)鍵步驟，受許多血管生成因子的調(diào)控。其中VEGF已被證實(shí)在膠質(zhì)瘤等多種惡性腫瘤的發(fā)生發(fā)展中起重要作用，其單克隆抗體貝伐單抗已成為一些惡性腫瘤的主要輔助治療手段，但仍有一定的局限性。Endocan于1996年首次被發(fā)現(xiàn)，由血管內(nèi)皮細(xì)胞分泌，是血管生成的重要調(diào)控分子。近年來，國(guó)內(nèi)外發(fā)表了諸多關(guān)于endocan作為腫瘤標(biāo)記的研究，相繼報(bào)道了人類不同組織中endocan表達(dá)及其與腫瘤惡性程度的關(guān)系，指出乳頭狀癌、腎透明細(xì)胞癌、肝癌、胃癌、大腸癌等腫瘤組織中endocan表達(dá)量明顯高于正常對(duì)照組，且endocan在腫瘤組織中的高表達(dá)與預(yù)后不良密切相關(guān)[9,17-20]。國(guó)內(nèi)外大量研究充分表明，endocan介導(dǎo)新生血管形成和癌癥進(jìn)展，可能是腫瘤發(fā)生發(fā)展的一個(gè)促進(jìn)因素。HCMV參與膠質(zhì)瘤發(fā)生發(fā)展的過程可能是復(fù)雜的，尚需進(jìn)一步深入闡明。但更多的研究認(rèn)為，病毒蛋白通過與宿主相互作用，引發(fā)炎癥和激活關(guān)鍵的信號(hào)通路，可能在腫瘤發(fā)生中起重要作用。本研究發(fā)現(xiàn)， HCMV 感染能通過激活STAT3上調(diào)膠質(zhì)瘤細(xì)胞中endocan表達(dá)，但有關(guān)HCMV參與膠質(zhì)瘤發(fā)生的機(jī)制尚待進(jìn)一步研究。

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s. AN Jing, E-mail: anjing@ccmu.edu.cn; WANG Yisong, E-mail: yswang@ccmu.edu.cn

Human cytomegalovirus infection is involved in STAT3-upregulated endocan expression in gliomas

WANG Shijie, FAN Dongying, LUO Qiaoli, XING Yan, WANG Yisong, AN Jing

DepartmentofMicrobiology,SchoolofBasicMedicalSciences,CapitalMedicalUniversity,Beijing100069,China

The aim of this study is to investigate the role of human cytomegalovirus (HCMV) infection in glioma pathogenesis and its possible mechanism. Based on the previous experiments, the expressions of signal transducer and activator of transcription 3 (STAT3) and endocan in HCMV-infected U87 cells, a human glioma cell line, were detected, and the relationship among HCMV, STAT3 and endocan was analyzed. The results showed that HCMV infection induced the upregulation of endocan mRNA and protein as well as the activated STAT3 in U87 cells. On the other hand, decreased levels of endocan mRNA and protein were observed in U87-STAT3-knockdown cells by RNAi. Furthermore, downregulated levels of endocan mRNA and protein were found in U87 cells treated by ganciclovir, an anti-viral drug. Then the expression of pSTAT3 in 79 glioma specimens and 8 control brain tissues was detected by immunohistochemistry (IHC) and the relationship between pSTAT3 level and glioma grade was analyzed. Compared with the control brain samples, increased level of pSTAT3 was found in glioma specimens and the staining intensity was related to glioma grade. The results suggest that HCMV infection is involved in glioma pathogenesis via upregulating endocan expression through STAT3 signaling pathway, and further provide some new clues for the treatment of glioma.

Glioma; Human cytomegalovirus; Endocan; Signal transducer and activator of transcription 3

國(guó)家自然科學(xué)基金(81271839、81471957)，北京市教育委員會(huì)科技計(jì)劃一般項(xiàng)目(KM201610025001)，第48批教育部留學(xué)回國(guó)人員科研啟動(dòng)基金，首都醫(yī)科大學(xué)自然基金(2015ZR11)

安靜，王一松

2016-08-10)