李洪利，尹崇高

(濰坊醫(yī)學(xué)院 1.醫(yī)學(xué)研究實(shí)驗(yàn)中心; 2.護(hù)理學(xué)院，山東 濰坊 261053)

研究論文

PTTG1通過上皮-間質(zhì)轉(zhuǎn)化促進(jìn)非小細(xì)胞肺癌的遷移和侵襲

李洪利1，尹崇高2*

(濰坊醫(yī)學(xué)院 1.醫(yī)學(xué)研究實(shí)驗(yàn)中心; 2.護(hù)理學(xué)院，山東 濰坊 261053)

目的研究PTTG1在非小細(xì)胞肺癌侵襲轉(zhuǎn)移中的作用。方法應(yīng)用免疫組織化學(xué)染色法檢測80例NSCLC組織中PTTG1、E-cadherin和Vimentin的表達(dá)，并分析其相關(guān)性。細(xì)胞分組：1)A549/con，轉(zhuǎn)染空載的A549，2)A549/PTTG1，轉(zhuǎn)染目的基因的A549，3)Scr/H1299，轉(zhuǎn)染空載的H1299，4)SiPTTG1/H1299，敲除PTTG1的H1299；用Western blot檢測細(xì)胞中PTTG1、E-cadherin、Vimentin蛋白的表達(dá)情況。結(jié)果NSCLC組織中PTTG1蛋白的陽性表達(dá)率為85%與E-cadherin的表達(dá)呈負(fù)相關(guān)(Plt;0.05)而與Vimentin的表達(dá)呈正相關(guān)(Plt;0.05)。Western blot結(jié)果顯示在SiPTTG1/H1299細(xì)胞中PTTG1蛋白表達(dá)水平明顯降低，Vimentin蛋白表達(dá)水平降低而E-cadherin蛋白表達(dá)水平升高。同時(shí)在A549/PTTG1細(xì)胞中Vimentin蛋白表達(dá)水平升高而E-cadherin蛋白表達(dá)水平降低。結(jié)論P(yáng)TTG1能促進(jìn)NSCLC EMT的發(fā)生，從而促進(jìn)NSCLC的侵襲和轉(zhuǎn)移。

垂體腫瘤轉(zhuǎn)化基因1；上皮-間質(zhì)轉(zhuǎn)化；肺腫瘤；侵襲

垂體腫瘤轉(zhuǎn)化基因(pituitary tumor-transforming gene,PTTG)被證實(shí)與細(xì)胞增殖、分化和凋亡有關(guān)的癌基因，其家族包括PTTG1、PTTG2和PTTG3 3個(gè)成員。PTTG1是研究最多的，因此通常被稱為PTTG[1]。本研究先前已證實(shí)PTTG1在NSCLC中的過度表達(dá)與淋巴結(jié)轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移有關(guān)，并可以通過多種機(jī)制影響NSCLC細(xì)胞的侵襲和轉(zhuǎn)移并且其表達(dá)受miR-186調(diào)控[2]。而腫瘤的侵襲和轉(zhuǎn)移的機(jī)制很多，PTTG1是否還能通過其他的分子機(jī)制影響NSCLC的轉(zhuǎn)移有待于進(jìn)一步的研究。

上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition, EMT)是指在某些生理或病理?xiàng)l件下，上皮細(xì)胞喪失細(xì)胞極性而獲得間葉細(xì)胞特性的過程[3]。更多的研究已經(jīng)表明EMT與許多腫瘤的侵襲和轉(zhuǎn)移是相關(guān)的。那么，PTTG1是否也可以通過EMT從而影響NSCLC的侵襲和轉(zhuǎn)移呢？本研究應(yīng)從組織和細(xì)胞兩個(gè)方面探討PTTG1在NSCLC侵襲轉(zhuǎn)移中的作用。

1 材料與方法

1.1 材料

1.1.1 實(shí)驗(yàn)標(biāo)本：收集80例濰坊醫(yī)學(xué)院附屬醫(yī)院手術(shù)切除NSCLC組織。所有病例經(jīng)病理證實(shí)，患者術(shù)前均未接受放療、化療和免疫治療。所有標(biāo)本都取得了患者的知情同意。

1.1.2 主要試劑：PTTG1、E-cadherin、vimentin一抗(Santa Cruz公司)；小RNA干擾載體(上海吉瑪制藥技術(shù)有限公司構(gòu)建)；pcDNA3.1-PTTG1載體(濰坊醫(yī)學(xué)院醫(yī)學(xué)研究實(shí)驗(yàn)中心)。

1.2 方法

1.2.1 免疫組織化學(xué)染色：染色方法，組織陽性結(jié)果判斷參照參考文獻(xiàn)[2]。

1.2.2 細(xì)胞培養(yǎng)及分組：H1299細(xì)胞常規(guī)培養(yǎng)于含10%胎牛血清的1640培養(yǎng)基中傳代培養(yǎng)。細(xì)胞分為4組：1)瞬時(shí)轉(zhuǎn)染插入空載質(zhì)粒的H1299細(xì)胞稱為Scr/H1299細(xì)胞；2)瞬時(shí)轉(zhuǎn)染插入PTTG1目標(biāo)片段5′-GAGAAGACUGUUAAAGCAATT-3′的H1299細(xì)胞稱為SiPTTG1/H1299細(xì)胞。3)構(gòu)建載體pcDNA3.1-PTTG1轉(zhuǎn)染pcDNA3.1-PTTG1的A549細(xì)胞稱為A549/PTTG1細(xì)胞；4)轉(zhuǎn)染空載pcDNA3.1的A549細(xì)胞稱為A549/con細(xì)胞。

1.2.3 Western blot檢測各組細(xì)胞培養(yǎng)72 h后提取總蛋白。各組以等量蛋白被12%SDS-PAGE電泳分離后，按200 mA，2 h的條件濕轉(zhuǎn)至PVDF膜上，封閉后加入相應(yīng)一抗和二抗，用ECL發(fā)光劑顯影，X膠片曝光。

1.3 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1PTTG1、E-cadherin及vimentin在NSCLC表達(dá)的相關(guān)性分析

陽性染色顆粒呈棕黃色或棕褐色。PTTG1和vimentin定位于腫瘤細(xì)胞的胞質(zhì)，而E-cadherin定位于腫瘤細(xì)胞的細(xì)胞膜。NSCLC組織中PTTG1的陽性表達(dá)率為85%(68/80)，E-cadherin的陽性表達(dá)率為66.2%(53/80)，vimentin的陽性表達(dá)率為36.2(29/80)。PTTG1在NSCLC中的表達(dá)與E-cadherin的表達(dá)呈負(fù)相關(guān)而與vimentin的表達(dá)呈正相關(guān)(Plt;0.05)(表1，圖1)。

表1 PTTG1與E-cadherin及vimentin在NSCLC表達(dá)的相關(guān)性

*Plt;0.05 compared with negative expression of PTTG1.

2.2Westernblot檢測肺癌組織及細(xì)胞系A(chǔ)549、H1299中PTTG1的表達(dá)

PTTG1蛋白質(zhì)在NSCLC組織中的表達(dá)明顯高于癌旁正常肺組織(圖2A)。此外，PTTG1蛋白在高度侵襲性細(xì)胞H1299 中表達(dá)高而在低侵襲性的A549細(xì)胞中表達(dá)較低(圖2B)。

A.positive expression of PTTG1 in lung adenocarcinoma; B.negative expression of E-cadherin in lung adenocarcinoma; C.positive expression of vimentin in lung adenocarcinoma; D.negative expression of PTTG1 in lung adenocarcinoma; E.positive expression of E-cadherin in lung adenocarcinoma; F.negative expression of vimentin in lung adenocarcinoma; G.positive expression of PTTG1 in lung squamous cell carcinoma; H.negative expression of E-cadherin in lung squamous cell carcinoma; I.positive expression of vimentin in lung squamous cell carcinoma

圖1免疫組織化學(xué)方法檢測非小細(xì)胞肺癌組織中PTTG1,E-cadherin及vimentin的表達(dá)
Fig1ExpressionofPTTG1,E-cadherinandvimentininNSCLCtissuebyimmunohistochemicalstaining(×100)

A.expression of PTTG1 in NSCLC patients; B.expression of PTTG1 in NSCLC cell lines圖2 PTTG1在NSCLC患者及細(xì)胞系中的表達(dá)Fig 2 Expression of PTTG1 in NSCLC patients andNSCLC cell lines

2.3已轉(zhuǎn)染NSCLC細(xì)胞系中PTTG1、E-cadherin、vimentin蛋白的表達(dá)

Western blot結(jié)果顯示，PTTG1蛋白的表達(dá)量在SiPTTG1/H1299細(xì)胞中較Scr/H1299細(xì)胞中明顯減少，而在A549/PTTG1細(xì)胞中較A549/con細(xì)胞中明顯增多，提示細(xì)胞轉(zhuǎn)染成功。Vimentin蛋白在SiPTTG1/H1299細(xì)胞(0.26±0.09)中的表達(dá)量較Scr/H1299細(xì)胞(1.29±0.14)中明顯減少(Plt;0.001)，而在A549/PTTG1細(xì)胞(0.84±0.08)中表達(dá)量比A549/con細(xì)胞(0.16±0.09)明顯增多(Plt;0.001)。E-cadherin蛋白在SiPTTG1/H1299細(xì)胞(0.86±0.09)中的表達(dá)量較Scr/H1299細(xì)胞(0.17±0.07)中明顯增多(Plt;0.001)，而在A549/PTTG1細(xì)胞(0.46±0.11)中表達(dá)量比A549/con細(xì)胞(0.94±0.09)中明顯減少(Plt;0.001)(圖3)。結(jié)果表明PTTG1可以明顯促進(jìn)NSCLC細(xì)胞的EMT發(fā)生。

圖3 Western blot檢測PTTG1, E-cadherin及vimentin在轉(zhuǎn)染細(xì)胞中的表達(dá)Fig 3 Expression of PTTG1, E-cadherin and vimentinin transfected cells by Western blot

2.4 已轉(zhuǎn)染NSCLC細(xì)胞系的形態(tài)學(xué)觀察

光學(xué)顯微鏡下觀察各組細(xì)胞的形態(tài)學(xué)變化(圖4)，結(jié)果發(fā)現(xiàn)A549/PTTG1細(xì)胞與A549/con細(xì)胞相比，細(xì)胞變得細(xì)長偽足不同程度的增多；而SiPTTG1/H1299細(xì)胞與Scr/H1299細(xì)胞相比，細(xì)胞偽足減少。

圖4 各種細(xì)胞的形態(tài)學(xué)改變Fig 4 Morphological change of A549/PTTG1, A549/con,SiPTTG1/H1299 and Scr/H1299 cells(×200)

3 討論

PTTG1是一種腫瘤轉(zhuǎn)化基因，在沒有任何輔助基因的參與下就能引起細(xì)胞的轉(zhuǎn)化，其與許多腫瘤的發(fā)生存在密切的關(guān)系[4]。PTTG1已經(jīng)被認(rèn)定為癌基因。PTTG1的過度表達(dá)可以促進(jìn)細(xì)胞的增殖和促進(jìn)裸鼠的腫瘤生成[5]。PTTG1的表達(dá)水平與腫瘤生成、血管生成和轉(zhuǎn)移都有密切的關(guān)系[6- 7]。PTTG1可以通過調(diào)節(jié)Twist、Snail、Slug和E-cadherin從而促進(jìn)卵巢癌EMT的發(fā)生[8]。本研究先前結(jié)果表明PTTG1可以通過cofilin/LIMK的磷酸化促進(jìn)NSCLC細(xì)胞的F- actin的聚合和通過PI3K/Akt/MMP途徑促進(jìn)NSCLC的侵襲和轉(zhuǎn)移[2]。本實(shí)驗(yàn)應(yīng)用免疫組織化學(xué)的方法檢測了NSCLC組織中PTTG1、E-cadherin和vimentin的表達(dá)，結(jié)果顯示PTTG1與E-cadherin的表達(dá)呈負(fù)相關(guān)，而與vimentin的表達(dá)呈正相關(guān)。

許多研究表明，在胚胎發(fā)育過程中，參與EMT的基因同時(shí)也可以控制細(xì)胞的轉(zhuǎn)移過程，EMT是多種腫瘤發(fā)生轉(zhuǎn)移的一個(gè)很重要的原因[9]。E-cadherin在多種腫瘤組織如肺癌、結(jié)腸癌等表達(dá)下調(diào)，其表達(dá)下調(diào)與腫瘤的復(fù)發(fā)和轉(zhuǎn)移密切相關(guān)[10]。腫瘤細(xì)胞發(fā)生EMT的重要標(biāo)志是E-cadherin表達(dá)下調(diào)。Vimentin在正常的上皮細(xì)胞中沒有表達(dá)，是間葉細(xì)胞的標(biāo)志物[11]。EMT的另一個(gè)重要標(biāo)志是上皮細(xì)胞表達(dá)vimentin[12]。本實(shí)驗(yàn)應(yīng)用兩種NSCLC細(xì)胞系從正反兩個(gè)方面檢測了PTTG1在NSCLC EMT中的作用，結(jié)果顯示PTTG1可以促進(jìn)低侵襲性細(xì)胞系A(chǔ)549發(fā)生EMT從而促進(jìn)A549的侵襲和轉(zhuǎn)移，而敲除高侵襲性細(xì)胞系H1299中的PTTG1可以反轉(zhuǎn)EMT的發(fā)生從而阻止H1299細(xì)胞的侵襲和轉(zhuǎn)移。

綜上所述，PTTG1能促進(jìn)NSCLC EMT的發(fā)生，從而促進(jìn)NSCLC的浸潤和轉(zhuǎn)移。此實(shí)驗(yàn)結(jié)果表明，PTTG1的抑制可以作為反轉(zhuǎn)EMT的新靶點(diǎn)從而抑制NSCLC的浸潤和轉(zhuǎn)移，為提高NSCLC患者的生存率提供實(shí)驗(yàn)和理論依據(jù)。

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PTTG1 induces migration and invasion ofnon-small cell lung cancer through epithelial-mesenchymal transition

LI Hong-li1, YIN Chong-gao2*

(1.Medicine Research Center; 2.College of Nursing, Weifang Medical University，Weifang 261053, China)

ObjectiveTo investigate the role of PTTG1 in the migration and invasion of Non-small cell lung cancer.MethodsTo detect the expression of PTTG1, E-cadherin and Vimentin in 80 cases of NSCLC using immunohistochemistry. Cells were divided into four groups: 1)A549/con, cells were transfected by empty vector; 2)A549/PTTG1, cells were transfected by purpose gene; 3)Scr/H1299, cells were transfected by empty vector; 4)SiPTTG1/H1299, cells were knocked down purpose gene. Western blot was used to analyze the expression of PTTG1, E-cadherin and Vimentin.ResultsThe protein expression of PTTG1 in NSCLC was negatively correlated with E-cadherin and positively correlated with Vimentin (Plt;0.05). H1299 cells expressed higher PTTG1, and A549 cells expressed lower PTTG1. The expressions of PTTG1 and Vimentin were lower in SiPTTG1/H1299 cells than those in Scr/H1299 and H1299 cells after trancfected 24 hours, but the E-cadherin expression was higher. At the same time, the protein expression of Vimentin was up-regulated and the expression of E-cadherin was down-regulated in A549/PTTG1 cells.ConclusionsPTTG1 promotes migration and invasion of NSCLC through EMT.

PTTG1; EMT; Lung tumor; migration

2014- 02- 27

2014- 04- 23

山東省中青年科學(xué)家科研基金(BS2011YY060)；山東省高等學(xué)?？萍加?jì)劃(J10LF64，J12LK03，J13LK03)

*通信作者(correspondingauthor)：ycg0711@sina.com

1001-6325(2014)10-1376-05

R 737.9；R 73- 37

A