李寧寧　熊　佶　汪　寅　朱靜靜　劉　穎△

(1復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院病理學(xué)系　上?！?0032; 2 復(fù)旦大學(xué)附屬華山醫(yī)院病理科　上?！?00040)

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星形膠質(zhì)細(xì)胞瘤IDH1突變對(duì)TAZ蛋白表達(dá)的影響

李寧寧1熊佶2汪寅2朱靜靜2劉穎1△

(1復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院病理學(xué)系上海20032;2復(fù)旦大學(xué)附屬華山醫(yī)院病理科上海200040)

目的研究星形膠質(zhì)細(xì)胞瘤異檸檬酸脫氫酶1 (isocitrate dehydrogenase 1,IDH1)突變對(duì)TAZ (transcriptional coactivator with PDZ-binding motif)蛋白的影響,并探討相關(guān)機(jī)制。方法采用穩(wěn)定轉(zhuǎn)染突變型IDH1-132H (132H)及野生型IDH1-132R (WT)的膠質(zhì)母細(xì)胞瘤 (U87MG)細(xì)胞,通過Western blot法檢測(cè)細(xì)胞內(nèi)TAZ蛋白表達(dá);免疫組化法檢測(cè)14例IDH1突變陰性和7例IDH1突變陽性的人腦膠質(zhì)母細(xì)胞瘤組織樣本中TAZ蛋白及其胞質(zhì)結(jié)合蛋白14-3-3e的表達(dá)差異;qRT-PCR觀察兩組細(xì)胞TAZ在mRNA水平有無差異;Western blot方法檢測(cè)Hippo信號(hào)通路核心激酶LATS1及磷酸化TAZ蛋白的表達(dá),以及14-3-3e蛋白的表達(dá)。結(jié)果Western blot結(jié)果表明 IDH1突變的星形膠質(zhì)細(xì)胞瘤中TAZ蛋白表達(dá)降低;人體膠質(zhì)細(xì)胞瘤組織的免疫組織化學(xué)結(jié)果與Western blot一致,證實(shí)了IDH1突變的星形膠質(zhì)細(xì)胞瘤中TAZ胞漿結(jié)合蛋白14-3-3e表達(dá)升高;qRT-PCR發(fā)現(xiàn)IDH1突變細(xì)胞TAZ mRNA水平相比野生型細(xì)胞表達(dá)明顯降低;IDH1突變細(xì)胞中,LATS1以及TAZ磷酸化水平升高且細(xì)胞14-3-3e蛋白表達(dá)升高。結(jié)論星形膠質(zhì)細(xì)胞瘤IDH1突變導(dǎo)致TAZ mRNA及蛋白水平表達(dá)降低,并通過活化Hippo 信號(hào)通路影響TAZ蛋白的磷酸化水平從而影響TAZ表達(dá)。

星形膠質(zhì)細(xì)胞瘤;IDH1突變;TAZ蛋白;Hippo 信號(hào)通路;14-3-3e蛋白

膠質(zhì)細(xì)胞瘤是中樞神經(jīng)系統(tǒng)最常見的惡性腫瘤,而膠質(zhì)母細(xì)胞瘤 (glioblastoma,GBM)是其中侵襲性最高、預(yù)后最差的一種類型[1]。由于分子生物學(xué)技術(shù)的進(jìn)步,對(duì)腫瘤的診斷逐步向可靠、實(shí)用的分子診斷方向發(fā)展[2]。異檸檬酸脫氫酶1 (isocitrate dehydrogenase 1,IDH1)突變是星形膠質(zhì)細(xì)胞瘤診斷中一個(gè)非常關(guān)鍵且熱門的分子標(biāo)記物,在腫瘤中發(fā)現(xiàn)的IDH1-R132H突變,表現(xiàn)為編碼IDH1蛋白132位氨基酸由精氨酸變?yōu)榻M氨酸[3]。有研究統(tǒng)計(jì),R132H在Ⅱ～Ⅲ級(jí)膠質(zhì)瘤中的發(fā)生率約80%,在GBM患者中約12%,現(xiàn)普遍認(rèn)為其與腫瘤侵襲性及患者預(yù)后密切相關(guān)[4],但具體機(jī)制仍有待進(jìn)一步研究。

Hippo信號(hào)通路最早在果蠅中發(fā)現(xiàn),參與調(diào)控細(xì)胞的生長(zhǎng)、增殖、凋亡,它與器官大小和組織再生密切相關(guān)。當(dāng)遺傳或環(huán)境因素導(dǎo)致其調(diào)節(jié)機(jī)制失效,可使細(xì)胞數(shù)目增多、器官增大,從而導(dǎo)致腫瘤發(fā)生[5]。TAZ (transcriptional coactivator with PDZ-binding motif)蛋白和YAP (yes-associated protein)是同源蛋白,兩者都是Hippo信號(hào)通路下游的致癌性轉(zhuǎn)錄共激活因子[6],TAZ可在細(xì)胞核內(nèi)與轉(zhuǎn)錄因子結(jié)合調(diào)控下游基因表達(dá),進(jìn)而參與細(xì)胞內(nèi)的信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞增殖與凋亡以及組織器官穩(wěn)態(tài)的調(diào)控[7]。在TAZ入核發(fā)揮作用的過程中受到Hippo信號(hào)通路的負(fù)性調(diào)控,即TAZ受到Hippo信號(hào)通路尤其是激酶LATS的調(diào)控而磷酸化,磷酸化的TAZ可與其胞質(zhì)中的結(jié)合蛋白14-3-3結(jié)合而滯留在細(xì)胞質(zhì)中[8],使TAZ降解增多、活性降低[9]。有研究發(fā)現(xiàn)TAZ蛋白在膠質(zhì)瘤中呈高表達(dá),其表達(dá)情況與腫瘤侵襲性及腫瘤級(jí)別正相關(guān),且發(fā)現(xiàn)TAZ蛋白的表達(dá)與腫瘤預(yù)后呈負(fù)相關(guān),這提示TAZ可能在膠質(zhì)瘤的發(fā)生和發(fā)展中起重要作用[10]。腫瘤內(nèi)在的生物學(xué)特性,特別是分子水平的差異很可能與腫瘤的發(fā)生、發(fā)展及預(yù)后有密切關(guān)系。本研究首次將膠質(zhì)細(xì)胞瘤IDH1突變與TAZ蛋白的表達(dá)聯(lián)系起來,致力于發(fā)現(xiàn)膠質(zhì)細(xì)胞瘤發(fā)生IDH1突變后其生物學(xué)行為發(fā)生改變的分子機(jī)制,從而為臨床提供個(gè)體化治療的理論依據(jù)。

材 料 和 方 法

細(xì)胞培養(yǎng) 人膠質(zhì)母細(xì)胞瘤細(xì)胞系U87MG (美國ATCC公司),穩(wěn)定轉(zhuǎn)染突變型IDH1-132H突變及空載對(duì)照,分別標(biāo)記為IDH1-132H和IDH1-WT兩株細(xì)胞 (復(fù)旦大學(xué)IBS熊躍教授惠贈(zèng)),用含10%FBS (美國Gibco公司) 的DMEM高糖培養(yǎng)基 (美國Gibco公司)培養(yǎng),細(xì)胞培養(yǎng)在37 ℃恒溫培養(yǎng)箱中,培養(yǎng)室內(nèi)通入5% CO2以維持細(xì)胞培養(yǎng)液的酸堿度平衡。

臨床樣本樣本來自復(fù)旦大學(xué)附屬華山醫(yī)院神經(jīng)外科2012—2015年的手術(shù)石蠟組織,依據(jù)中樞神經(jīng)系統(tǒng)腫瘤診斷標(biāo)準(zhǔn) (Word Health Organization,2000)診斷為GBM,14例IDH1陰性,7例IDH1陽性 (IDH1-R132H特異性抗體,德國Dianova公司,internal clone H09,稀釋度1∶100),所有人體組織學(xué)樣本獲取及使用過程獲復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院倫理委員會(huì)批準(zhǔn)。

Western blot 運(yùn)用Western blot方法檢測(cè)132H及WT細(xì)胞中TAZ (TAZ兔抗,美國CST公司,貨號(hào)8418,稀釋度1∶1 000),磷酸化TAZ [兔抗P-TAZ (SER89),美國Santa Cruz公司,貨號(hào)sc-17610-R,稀釋度1∶200],LATS1 (兔抗LATS1,美國CST公司,貨號(hào)3477,稀釋度1∶1 000)以及14-3-3e (兔抗14-3-3e,美國CST公司,貨號(hào)9635,稀釋度1∶1 000)蛋白的表達(dá)。內(nèi)參選用GAPDH (鼠抗GAPDH,美國Santa Cruz公司,貨號(hào)sc-166574,稀釋度1∶2 000),二抗 (羊抗小鼠/兔二抗,美國Proteintech公司,貨號(hào)SA00001-1/2,稀釋度1∶1 000),將細(xì)胞裂解后提取細(xì)胞蛋白并定量,轉(zhuǎn)膜后與相應(yīng)蛋白抗體孵育過夜,第2天加二抗并顯色。

免疫組化TAZ單克隆抗體 (鼠抗TAZ,美國BD PharmingenTM公司,貨號(hào)560235,稀釋度1∶200),14-3-3e (兔抗14-3-3e,美國CST公司,貨號(hào)9635,稀釋度1∶1 000),人腦GBM石蠟組織樣本,采用辣根過氧化物酶法檢測(cè)TAZ蛋白的表達(dá)情況。

qRT-PCR運(yùn)用qRT-PCR法檢測(cè)132H和WT兩株細(xì)胞中TAZ mRNA的表達(dá)。將細(xì)胞PBS洗凈后加入預(yù)冷的Trizol提取RNA,逆轉(zhuǎn)錄成cDNA后進(jìn)行qRT-PCR。10 μL PCR體系包括:SYBR Premix EX Taq 5 μL,ROX Reference Dye 0.2 μL,上下游引物各0.2 μL,cDNA模板1 μL,ddH2O 3.4 μL。PCR反應(yīng)條件為:95 ℃ 40 s,95 ℃ 35 s,60 ℃ 1 min,循環(huán)40次。

統(tǒng)計(jì)學(xué)處理 Alphaview SA軟件對(duì)Western blot 的結(jié)果進(jìn)行灰度分析,Graphpad 6.1軟件對(duì)所有數(shù)據(jù)進(jìn)行統(tǒng)計(jì)處理統(tǒng)計(jì)處理。采用雙側(cè)t檢驗(yàn),P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)　　　果

TAZ蛋白在132H和WT細(xì)胞中的表達(dá)為了檢測(cè)星形膠質(zhì)細(xì)胞瘤IDH1突變與TAZ蛋白之間的關(guān)系,采用穩(wěn)定轉(zhuǎn)染突變型IDH1-132H及野生型IDH1-132R的膠質(zhì)母細(xì)胞瘤 (U87MG)細(xì)胞,通過Western blot 法檢測(cè)TAZ蛋白的表達(dá)情況。IDH1-R132H組細(xì)胞TAZ蛋白表達(dá)較IDH1-WT組低,差異有統(tǒng)計(jì)學(xué)意義 (圖1)。

人腦石蠟組織切片TAZ蛋白表達(dá)水平采用免疫組化法檢測(cè)TAZ蛋白及其胞質(zhì)結(jié)合蛋白14-3-3e在人體組織樣本中的表達(dá)情況。將TAZ免疫組化標(biāo)記結(jié)果按12分法進(jìn)行半定量分析。染色強(qiáng)度分為0～3分,陰性標(biāo)記為0分,弱陽性標(biāo)記為1分,中等強(qiáng)度標(biāo)記為2分,強(qiáng)陽性標(biāo)記為3分,陽性細(xì)胞百分比對(duì)應(yīng)值:將陽性的細(xì)胞數(shù)<5%算為0分,5%～25%為1分,26%～50%為2分,51%～75%為3分,>75%為4分。然后將染色強(qiáng)度×陽性細(xì)胞百分比對(duì)應(yīng)的值作為評(píng)分,最高分為12分。免疫組化結(jié)果顯示132H突變型GBM病例組TAZ蛋白表達(dá)較野生型GBM病例組明顯降低,而14-3-3e蛋白的表達(dá)則明顯升高,差異均具有統(tǒng)計(jì)意義 (圖2,放大倍數(shù)為10×40倍)。

The expression of TAZ protein decreased in IDH1 mutated cells (132H) compared with that of control cells (WT);A:Western blot;B:The ratio of gray value.(1)P<0.05.

圖1IDH1-132H和IDH1-WT兩株細(xì)胞中TAZ蛋白的表達(dá)

Fig 1The expression of TAZ protein in IDH1-132H and IDH1-WT cells

qRT-PCRqRT-PCR檢測(cè)兩株細(xì)胞中TAZ mRNA的表達(dá),以β-actin為內(nèi)參,RQ值采用2-Δct的方法計(jì)算,132H細(xì)胞TAZ在mRNA水平降低,差異有統(tǒng)計(jì)學(xué)意義 (圖3)。

Western blotTAZ最初是作為14-3-3蛋白的底物被發(fā)現(xiàn)的,其結(jié)合需要TAZ第89位絲氨酸的磷酸化,TAZser 89在YAP中對(duì)應(yīng)的相同位置是YAPser 127位點(diǎn),這兩個(gè)位點(diǎn)恰好符合14-3-3蛋白其中的一個(gè)最佳結(jié)合序列[11]。Hippo信號(hào)通路通過調(diào)節(jié)絲氨酸/蘇氨酸蛋白激酶LATS,使TAZ的第89位絲氨酸 (YAP的第127位絲氨酸)發(fā)生磷酸化,從而產(chǎn)生14-3-3蛋白的結(jié)合序列,TAZ與14-3-3e蛋白結(jié)合滯留胞質(zhì)內(nèi)或者直接降解,最終使TAZ活性及表達(dá)量均有所降低[12-13]。為了進(jìn)一步驗(yàn)證IDH1突變是否影響Hippo信號(hào)對(duì)TAZ的調(diào)控,我們采用Western blot檢測(cè)IDH1突變對(duì)Hippo信號(hào)通路活化狀態(tài)以及TAZ胞漿結(jié)合蛋白14-3-3e表達(dá)的影響。Western blot結(jié)果顯示在IDH1-132H細(xì)胞中LATS1表達(dá)高于WT細(xì)胞,差異有統(tǒng)計(jì)學(xué)意義;p-TAZ (ser89)、p-YAP (ser127)及14-3-3e蛋白表達(dá)明顯升高,差異均具有統(tǒng)計(jì)學(xué)意義 (圖4)。這一結(jié)果表明IDH1突變后,Hippo信號(hào)通路明顯活化,導(dǎo)致TAZ與胞質(zhì)中14-3-3e結(jié)合增多且降解增加。

A:HE staining of IDH1 mutated GBM samples (a) and wild type GBM samples (b);The expression of TAZ was decreased in IDH1 mutated samples (c) compared with that of control samples (d);on the contrary,the expression of 14-3-3e was increased in IDH1 mutated samples (e,f);B,C showed the IHC staining density of TAZ and 14-3-3e separately.(1)P<0.01;(2)P<0.05.

圖2人膠質(zhì)母細(xì)胞瘤組織中TAZ及14-3-3e免疫組化染色

Fig 2Immunohistochemistry staining of TAZ and 14-3-3e in human glioblastoma tissue

qRT-PCR showed that TAZ mRNA was also decreased after IDH1 mutation.(1)P<0.01.

圖3IDH1-132H和IDH1-WT兩株細(xì)胞TAZ mRNA表達(dá)

Fig 3The relative mRNA expression of TAZ in IDH1-132H and IDH1-WT cells

討　　　論

腦膠質(zhì)細(xì)胞瘤作為顱內(nèi)最常見的腫瘤,近年來發(fā)病率仍有上升的趨勢(shì)[14],腫瘤呈廣泛浸潤(rùn)性生長(zhǎng),單純手術(shù)難以完全切除,即使結(jié)合術(shù)后放化療,其術(shù)后復(fù)發(fā)率仍然很高,患者預(yù)后很差,生活質(zhì)量受到嚴(yán)重影響。2008年P(guān)arsons等[4]首次發(fā)現(xiàn)IDH1突變的存在,并發(fā)現(xiàn)IDH1突變?cè)谀z質(zhì)瘤的形成及診斷中發(fā)揮重要作用。IDH1突變也可見于急性髓系白血病 (acute myeloid leukemia,AML)、軟骨肉瘤等[15-16]。IDH1究竟以何種機(jī)制導(dǎo)致膠質(zhì)瘤的生物學(xué)行為發(fā)生改變?nèi)孕枰M(jìn)一步探索。

研究普遍認(rèn)為IDH1突變的高級(jí)別膠質(zhì)瘤相比于同級(jí)別的野生型侵襲性降低[17]。事實(shí)上,IDH1突變狀態(tài)能否作為高級(jí)別膠質(zhì)瘤的獨(dú)立預(yù)后因子仍存在很大爭(zhēng)議[3,18]。Hartmann等[19]的研究甚至表明,伴IDH1突變的Ⅳ級(jí)GBM預(yù)后比無IDH1突變的Ⅲ級(jí)間變性星形細(xì)胞瘤要好。因此,IDH1突變后膠質(zhì)瘤的生物學(xué)行為,包括腫瘤的侵襲性、轉(zhuǎn)移性以及預(yù)后仍然需要進(jìn)一步探索。

有研究表明TAZ作為一個(gè)原癌基因,可以促進(jìn)細(xì)胞增殖、遷移、轉(zhuǎn)化,從而促進(jìn)腫瘤的發(fā)生[20]。TAZ蛋白是Hippo信號(hào)通路下游最主要的效應(yīng)器,Hippo通路通過激活激酶LATS后直接使 TAZ發(fā)生磷酸化,產(chǎn)生14-3-3蛋白的結(jié)合位點(diǎn)。14-3-3蛋白分布于胞質(zhì)中,與TAZ結(jié)合后會(huì)促進(jìn)TAZ滯留在胞質(zhì),并且促進(jìn)其降解而失活[21]。本研究發(fā)現(xiàn)IDH1突變的膠質(zhì)瘤中TAZ蛋白低表達(dá),并在人膠質(zhì)瘤組織樣本中加以驗(yàn)證;本研究進(jìn)一步探討了TAZ蛋白表達(dá)降低的機(jī)制:首先發(fā)現(xiàn)TAZ在mRNA水平也有所降低,證明其轉(zhuǎn)錄水平的低表達(dá);同時(shí)也發(fā)現(xiàn)在IDH1突變的細(xì)胞中Hippo信號(hào)通路中的LATS表達(dá)有明顯改變,IDH1突變的細(xì)胞中TAZ的胞質(zhì)結(jié)合蛋白14-3-3e表達(dá)明顯升高。這些結(jié)果表明IDH1突變的星形膠質(zhì)細(xì)胞瘤通過Hippo信號(hào)通路的核心激酶影響TAZ與其胞漿結(jié)合蛋白的結(jié)合,最終影響TAZ蛋白表達(dá)。綜上所述,TAZ轉(zhuǎn)錄水平上低表達(dá)以及蛋白降解的增加導(dǎo)致了TAZ蛋白總量在IDH1突變的細(xì)胞中表達(dá)明顯下降。

The levels of P-TAZ (ser89),14-3-3e,LATS1 and P-YAP (127) were higher expressed in IDH1-132H cells than those in IDH1-WT cells (A) and the ratio of their gray values (B).(1)P<0.05.

圖4P-TAZ (ser89)、14-3-3e、 LATS1及P-YAP (127)蛋白的表達(dá)

Fig 4The expression levels of P-TAZ (ser89),14-3-3e,LATS1 and P-YAP (127) protein

目前證實(shí)IDH1突變可以產(chǎn)生兩種酶活性的改變:野生型IDH1功能的下降——即依賴NADP+的催化異檸檬酸轉(zhuǎn)化為α-KG (α-ketoglutarate)減少,以及新催化功能的獲得——即還原α-KG生成D-2-HG (D-2-Hydroxyglutarate)增多、同時(shí)伴有NADPH的氧化,最終導(dǎo)致α-KG水平下降,D-2-HG水平明顯升高[22-25],而正常人體其含量很低。現(xiàn)多認(rèn)為D-2-HG是一種腫瘤性代謝產(chǎn)物,由于D-2-HG與α-KG結(jié)構(gòu)的相似性,導(dǎo)致其可競(jìng)爭(zhēng)性抑制多種α-KG依賴的酶,從而引起人體代謝以及表觀遺傳學(xué)的改變[1]。哺乳動(dòng)物的Hippo信號(hào)通路受到精密調(diào)節(jié)并與許多信號(hào)通路之間存在密切聯(lián)系,從而共同調(diào)控細(xì)胞數(shù)量及器官大小。有研究發(fā)現(xiàn)Hippo信號(hào)通路與Wnt、CD44、shh、Notch[26-29]等信號(hào)通路密切相關(guān)。因此,IDH1突變后究竟以何種機(jī)制引發(fā)TAZ轉(zhuǎn)錄水平下降及Hippo信號(hào)通路的活化,尚待進(jìn)一步的研究。

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E-mail:yliu@shmu.edu.cn

Effects of mutated IDH1 on TAZ protein in human astrocytoma

LI Ning-ning1,XIONG Ji2,WANG Yin2,ZHU Jing-jing2,LIU Ying1△

(1DepartmentofPathology,SchoolofBasicMedicalSciences,FudanUniversity,Shanghai200032,China;2DepartmentofPathology,HuashanHospital,FudanUniversity,Shanghai200040,China)

ObjectiveTo investigate the effects of mutated isocitrate dehydrogenase 1 (IDHl) on TAZ (transcriptional coactivator with PDZ-binding motif protein) expression in human astrocytoma and to explore the relevant mechanisms.MethodsGlioblastoma (GBM) U87MG cells transfected with mutated IDH1-132H and wild type IDH1-132R (WT) were used to detected the TAZ protein expression by Western blot;The expression of TAZ protein and its cytoplasmic binding protein 14-3-3e were investigated in a cohort of GBM cases,which including 14 IDH1 wild type cases and 7 IDH1 mutated cases.Invitro,qRT-PCR was used to detect TAZ mRNA expression;Western blot was used to detect the levels of LATS1,phosphorylated TAZ and 14-3-3e protein.ResultsWe found that the expression of TAZ protein was decreased in IDH1 mutated cells compared with those of IDH1 wild type cells.This result was further verifiedinvivoby using immunohistochemistry to detect the expression of TAZ in human GBM samples with or without IDH1 mutation.Immunohistochemistry also showed that TAZ cytoplasmic binding protein 14-3-3e was increased;qRT-PCR showed that TAZ mRNA level was decreased in IDH1 mutated cells;and subsequent Western blot showed the levels of LATS1,TAZ phosphorylation and the 14-3-3e were increased in IDH1 mutant cells.ConclusionsIDH1 mutation in gliomacould reduce TAZ mRNA and its protein expression,increase the phosphorylation level of TAZ protein by activating Hippo signaling pathway,thereby furtherinhibit TAZ expression.

astrocytoma;IDH1 mutation;TAZ protein;Hippo signaling pathway;14-3-3e protein

R36, R739.4

Adoi: 10.3969/j.issn.1672-8467.2016.04.001

2016-02-19;編輯:王蔚)

國家自然科學(xué)基金面上項(xiàng)目(81272796)

*This work was supported by the General Program of National Natural Science Foundation of China (81272796).