SIA H2與P-ERK在乳腺癌中的表達(dá)及其臨床意義

孟 媛 董經(jīng)宇 甄 娟 孫 潔 宋 敏 李柏林

SIA H2與P-ERK在乳腺癌中的表達(dá)及其臨床意義

孟 媛 董經(jīng)宇3甄 娟 孫 潔 宋 敏1李柏林2＊

(中國醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院病理學(xué)教研室;1中國醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院病理學(xué)教研室及中國醫(yī)科大學(xué)附屬第一醫(yī)院病理科;2中國醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院化學(xué)教研室;3中國醫(yī)科大學(xué)92期七年制遼寧沈陽110001)

目的 觀察人乳腺細(xì)胞系及乳腺組織中SIAH2和P-ERK表達(dá)的變化,探討乳腺癌中SIAH2與P-ERK的關(guān)聯(lián)。方法 應(yīng)用免疫組織化學(xué)檢測(cè)140例乳腺石蠟包埋組織中SIAH2和P-ERK的表達(dá),Western blot檢測(cè)人乳腺細(xì)胞系和23例乳腺浸潤性導(dǎo)管癌及癌旁正常乳腺組織中SIA H2和P-ERK的蛋白表達(dá)情況,人乳腺癌細(xì)胞系表達(dá)SIA H2-siRNA后,P-ERK蛋白表達(dá)情況。結(jié)果 乳腺癌中SIAH2和P-ERK陽性率與組織學(xué)分級(jí)相關(guān)(P<0.05),SIAH2和P-ERK呈正相關(guān)性。人乳腺癌細(xì)胞系以及乳腺癌組織中SIAH2和P-ERK蛋白表達(dá)量明顯高于人乳腺正常上皮細(xì)胞系與癌旁正常乳腺組織中SIA H2和P-ERK蛋白表達(dá)量 (P<0.05);表達(dá)SIAH2-siRNA的乳腺癌細(xì)胞系與對(duì)照組相比,P-ERK蛋白表達(dá)明顯減少(P<0.05)。結(jié)論 SIA H2、P-ERK過表達(dá)與乳腺癌的組織學(xué)分級(jí)有關(guān)。在乳腺癌細(xì)胞系中抑制SIA H2表達(dá)后,P-ERK表達(dá)也減少,因此抑制SIA H2可以抑制 ERK通路。

SIAH2; P-ERK; 乳腺癌; 臨床病理特征; 干擾

乳腺癌是危害女性身體健康的惡性腫瘤之一,乳腺癌發(fā)生發(fā)展與癌細(xì)胞異常增殖相關(guān),所以促進(jìn)乳腺癌發(fā)生和生長的相關(guān)因子一直是分子生物學(xué)的研究重點(diǎn)。SIA H2(seven in absentia homologues)是果蠅SINA(seven in absentia)同系物,是一種 E3泛素連接酶,目前的研究已經(jīng)證實(shí)SIA H2在多種癌中表達(dá)都有異常。絲裂原活化蛋白激酶(mitogen activated protein kinase MAPK)途徑中的細(xì)胞外信號(hào)激酶(extracellular signal regulated kinase,ERK)是MAPK家族的重要成員,P-ERK是ERK的活性形式,ERK信號(hào)途徑因?yàn)槟軌蛞哉哉{(diào)節(jié)的方式調(diào)控腫瘤生長而備受關(guān)注。已有研究表明SIA H2與P-ERK可能有關(guān)聯(lián),那么在乳腺癌的發(fā)生發(fā)展中SIA H2是否與P-ERK具有關(guān)聯(lián),是否SIA H2可以通過調(diào)節(jié)活化的 ERK途徑發(fā)揮作用,這對(duì)指導(dǎo)乳腺癌的臨床診斷和治療具有重要臨床意義。本研究應(yīng)用免疫組織化學(xué)和Western blot檢測(cè)P-ERK與SIA H2在乳腺組織中和乳腺細(xì)胞系的表達(dá)情況,兩者的表達(dá)是否具有相關(guān)性,并進(jìn)一步研究SIA H2和P-ERK表達(dá)與臨床病理的聯(lián)系,評(píng)價(jià)SIA H2是否可能通過 ERK通路調(diào)節(jié)乳腺癌細(xì)胞生長,為 SIA H2小分子抑制劑應(yīng)用于乳腺癌治療提供相應(yīng)的理論依據(jù),為乳腺癌治療開辟新途徑。

材料和方法

1.臨床資料

選擇中國醫(yī)科大學(xué)附屬第一臨床醫(yī)院病理科140例乳腺患者手術(shù)切除腫物的存檔蠟塊(2007年12月至2009年6月)?；颊呔鶠榕?年齡24～71歲。所有患者術(shù)前均未做放化療以及免疫治療,有完整的病歷資料。依據(jù)WHO 2003年的乳腺腫瘤組織學(xué)分類標(biāo)準(zhǔn)分類,乳腺浸潤性導(dǎo)管癌伴淋巴結(jié)轉(zhuǎn)移38例,乳腺浸潤性導(dǎo)管癌淋巴結(jié)無轉(zhuǎn)移38例,乳腺導(dǎo)管原位癌44例,乳腺正常上皮20例。Western blot檢測(cè)所用的23例乳腺浸潤性導(dǎo)管癌(包括76例乳腺浸潤性導(dǎo)管癌)及癌旁正常新鮮乳腺組織,切除后立即置于-70℃冰凍保存。

2.材料

人乳腺正常上皮細(xì)胞系MCF-10A,和人乳腺癌細(xì)胞系MCF-7,MDA-MB-435S為本實(shí)驗(yàn)室儲(chǔ)備,SIA H2的特異性siRNA(sc-37497)和SIA H2的非特異性 siRNA(control siRNA序列)(sc-5507)購自Santa Cruz公司。

3.主要試劑

鼠抗人SIA H2和P-ERK單克隆抗體,兔抗人ERK多克隆抗體,購自Santa Cruz公司。S-P超敏免疫組化試劑盒(KIT29710)和DAB酶底物顯色試劑盒(DAB20030)均購自福州邁新生物技術(shù)開發(fā)公司。Western blot用,兔抗人多克隆抗β-actin抗體,羊抗兔和兔抗鼠二抗購自北京中杉金橋生物技術(shù)有限公司。DM EM購自美國 GIBCO。脂質(zhì)體Lipofectamine2000購自美國Invitrogen公司。

4.方法

4.1 細(xì)胞培養(yǎng)及SIA H2-siRNA干擾

MCF-10A,MCF-7,MDA-MB-435S細(xì)胞均采用DMEM(含有100 UPml青霉素,100 UPml硫酸鏈霉素和10%胎牛血清)培養(yǎng)液,0.1%胰酶消化傳代,在37℃、5%CO2培養(yǎng)箱中培養(yǎng)。實(shí)驗(yàn)時(shí),細(xì)胞處于對(duì)數(shù)生長期且細(xì)胞活力良好。在預(yù)實(shí)驗(yàn)中已經(jīng)確定SIA H2的 siRNA(sc-37497)可以有效干擾SIA H2。根據(jù)脂質(zhì)體 Lipofectamine2000的說明書,將 SIA H2-siRNA瞬時(shí)轉(zhuǎn)染至 MDA-MB-435S細(xì)胞中。

4.2 免疫組織化學(xué)S-P法

按S-P法試劑盒說明書步驟進(jìn)行 P-ERK(1:150)、SIA H2(1:200)抗體免疫組織化學(xué)染色。PBS代替一抗作為陰性對(duì)照,利用已知陽性片作為陽性對(duì)照。陽性判定:以黃色或棕黃色顆粒染色為陽性反應(yīng),P-ERK陽性表達(dá)于細(xì)胞漿和細(xì)胞核,SIA H2陽性表達(dá)于細(xì)胞核。參照溫媛媛等[1]報(bào)道的方法作為免疫組織化學(xué)陽性判斷標(biāo)準(zhǔn),即每張切片在光鏡下隨機(jī)選取5個(gè)高倍視野(400倍),每個(gè)視野計(jì)數(shù)100個(gè)瘤細(xì)胞。根據(jù)陽性細(xì)胞百分率,無陽性細(xì)胞為0分,陽性細(xì)胞1%-50%為1分,陽性細(xì)胞51%-75%為2分,陽性細(xì)胞≥75%為3分;按染色強(qiáng)度:1分呈淺黃色,2分呈黃色,3分呈棕黃色。陽性細(xì)胞數(shù)評(píng)分與染色強(qiáng)度評(píng)分相乘后得出綜合評(píng)分,綜合評(píng)分≥4為陽性表達(dá),<4為陰性。

4.3 Western blot

組織標(biāo)本處理是,4℃條件下取-70℃冰凍保存1～2g乳腺癌及癌旁正常組織標(biāo)本加入約5倍濕重的蛋白裂解緩沖液再充分剪碎,經(jīng)超聲勻漿粉碎后,4℃靜置一小時(shí);細(xì)胞標(biāo)本處理是,取對(duì)數(shù)生長期的細(xì)胞,加入細(xì)胞裂解液后,4℃下靜置1h。組織和細(xì)胞標(biāo)本都在低溫高速離心(4℃,12000 r/min,30 min),提取上清即為總蛋白。經(jīng) 12%的 SDSPA GE電泳、轉(zhuǎn)印,脫脂奶粉封閉,抗β-actin(1:500)和抗 P-ERK(1∶400)抗 SIA H2(1∶400)抗ERK(1∶400)4℃下孵育,過夜;二抗室溫下孵育2 h,ECL顯色,經(jīng)自動(dòng)電泳凝膠成像分析儀采集圖像。實(shí)驗(yàn)重復(fù)三次。

5.統(tǒng)計(jì)學(xué)分析

采用 SPSS17.0統(tǒng)計(jì)學(xué)軟件,進(jìn)行 Pearsonχ2檢驗(yàn),Spearman’s correlation檢驗(yàn),Fisher’S精確概率法。P<0.05有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1.SIA H2和P-ERK在乳腺組織中的表達(dá)

1.1 SIA H2陽性表達(dá)于細(xì)胞核,SIA H2在乳腺正常導(dǎo)管上皮(15.0%)、乳腺導(dǎo)管原位癌(56.8%)、乳腺浸潤性導(dǎo)管癌(69.7%)中陽性率呈遞增趨勢(shì)(表1),統(tǒng)計(jì)學(xué)分析結(jié)果為SIA H2在乳腺正常導(dǎo)管上皮與乳腺原位癌表達(dá)陽性率具有差異(P=0.002),SIA H2在乳腺正常導(dǎo)管上皮與乳腺浸潤性導(dǎo)管癌表達(dá)陽性率也具有明顯差異(P<0.001)。P-ERK陽性表達(dá)于細(xì)胞漿和細(xì)胞核,P-ERK在乳腺正常導(dǎo)管上皮(25.0%)、乳腺導(dǎo)管原位癌(52.3%)、乳腺浸潤性導(dǎo)管癌(75.0%)中陽性率呈遞增趨勢(shì)(表1);P-ERK蛋白在乳腺正常導(dǎo)管上皮,與原位癌和浸潤性導(dǎo)管癌表達(dá)都具有差異(P=0.041,P<0.001)(表 1,圖 1)。

圖1 SIA H2和P-ERK在乳腺癌中的表達(dá) 免疫組化S-P法×400 NBT,正常乳腺上皮;DCIS,乳腺導(dǎo)管內(nèi)癌;IDC,乳腺浸潤性導(dǎo)管癌。Fig.1 The expression of SIAH2 and P-ERK in breast tissue.S-P method×400 NBT,normal breast tissues;DCIS ductal carcinoma in situ;IDCinvasive ductal carcinoma

表1 SIA H2和P-ERK在140例乳腺組織中的表達(dá)Table 1 The Expression of SIA H2 and P-ERK in 140 Cases of Breast Tissues

1.2 SIA H2和P-ERK在乳腺癌組織中蛋白表達(dá)明顯高于配對(duì)的癌旁正常乳腺組織,且SIA H2和P-ERK在乳腺癌和癌旁正常乳腺組織的表達(dá)差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)(圖2)。

2.SIA H2和P-ERK在乳腺癌中的關(guān)系

76例乳腺癌中SIA H2和 P-ERK表達(dá)進(jìn)行相關(guān)分析,經(jīng)spearman相關(guān)性檢驗(yàn),結(jié)果顯示SIA H2和P-ERK表達(dá)呈正相關(guān)(rs=0.480,P<0.001)(表2)。

圖2 SIAH2和P-ERK在乳腺癌和癌旁正常乳腺組織中蛋白的表達(dá)N,癌旁正常乳腺組織;C,乳腺癌組織;2A乳腺浸潤性導(dǎo)管癌和癌旁正常組織中SIA H2和P-ERK表達(dá)情況;2B SIA H2和P-ERK與β-actin相比后蛋白相對(duì)表達(dá)量。Fig.2 The expression of SIAH2 and P-ERK in in breast cancer tissues and the adjacent normal breast tissues.N,adjacent normal breast tissues;C,breast cancer tissues;2A The expression of SIA H2 and P-ERK in breast cancer tissues and adjacent normal breast tissues;2B The relative protein expression of P-ERK and SIA H2 compared withβ-actin.bar,SD.

表2 SIA H2和P-ERK在76例乳腺癌組織中的關(guān)系Table 2 Relationships Between the Expression of SIA H1 and P-ERK in76 Cases of Breast Cancer Tissues

3.SIA H2和P-ERK在乳腺癌中的表達(dá)與臨床病理特征之間的關(guān)系

76例乳腺浸潤性癌中SIA H2和 P-ERK陽性率與組織學(xué)分級(jí)相關(guān)且組織學(xué)分級(jí)越低,SIA H2和P-ERK(SIA H2,P=0.019;P-ERK,P=0.005)陽性表達(dá)率越高(表3)。SIA H2在ER或PgR高表達(dá)時(shí)陽性率高(P值都小于0.001)(表3)。

表3 SIA H2和P-ERK在76例乳腺癌中的表達(dá)與臨床病理特征的關(guān)系Table 3 Relationship between SIA H2 and P-ERK protein expression and clinicopathological facors in invasive ductal carcinoma

4.SIA H2和P-ERK在乳腺細(xì)胞系中的表達(dá)

SIA H2和 P-ERK在MCF-10A中蛋白表達(dá)量明顯低于在MCF-7和MDA-MB-435S蛋白表達(dá)量,且具有統(tǒng)計(jì)學(xué)意義(P<0.05)。SIA H2在細(xì)胞系MDA-MB-435S中蛋白表達(dá)高于在MCF-7細(xì)胞系蛋白表達(dá)且具有統(tǒng)計(jì)學(xué)意義(P<0.05)(圖3)。

圖3 SIAH2和P-ERK在乳腺細(xì)胞系中蛋白表達(dá)3A MCF-10A,MCF-7,MDA-MB-435S中SIAH2和 P-ERK表達(dá)情況。3B SIAH2和 P-ERK與β-actin相比后蛋白相對(duì)表達(dá)量。Fig.3 The expression of SIAH2 and P-ERK in breast cell lines3A The expression of SIAH2 and P-ERK in MCF-10A MCF-7,MDA-MB-435S;3B The relative protein expression of PERK and SIAH2 compared withβ-actin.bar,SD.

5.蛋白轉(zhuǎn)染SIA H2-siRNA的MDA-MB-435S細(xì)胞系中P-ERK表達(dá)

在人乳腺癌細(xì)胞系 MDA-MB-435S表達(dá) SIA H2-siRNA后,P-ERK蛋白表達(dá)明顯減少,且具有統(tǒng)計(jì)學(xué)意義(P<0.05)(圖4)。

圖4 在MDA-MB-435S細(xì)胞系中表達(dá)SIAH2-siRNA后,P-ERK表達(dá)減少4A MDA-MB-435S表達(dá)control-RNA和SIA H2-siRNA后P-ERK表達(dá)情況;4B SIA H2和P-ERK與β-actin相比后蛋白相對(duì)表達(dá)量Fig.4 The expression of P-ERK in human breast cancer cell lines MDA-MB-435Swas downregulated by SIA H2-siRNA.4A The P-ERK levels were decreased in MDA-MB-435S cell transfected with SIA H2-siRNA compared with the cells transfected with control-siRNA or untreated;4B The difference of P-ERK expression in MDA-MB-435Scells transfected with SIAH2-siRNA(P<0.05),control-siRNA and untreated were statistically significant.bar,SD.

討 論

人類SIA H(seven in absentia homolog)基因包括 SIA H1和 SIA H2[2-3]二者是高度保守,且 SIA H與果蠅屬 SINA是同系物,SIA H2與 SINA具有68%的一致性[4]。SIA H2編碼325個(gè)氨基酸,SIA H1編碼282個(gè)氨基酸,SIA H2比SIA H1具有更長的N-末端,SIA H2與 SIA H1具有85%的一致性[3]。SIA H2的N-端是一個(gè)包含2個(gè)環(huán)域結(jié)構(gòu)的催化區(qū)域,C-端是高度保守的底物結(jié)合區(qū)(SBD)[2,5]。具有環(huán)域結(jié)構(gòu)的SIA H2是 E3泛素連接酶中的一員,它可以促進(jìn)與其相互連接的蛋白OBF-1[6],N-Co R[7],TRAF2[8],BA G-1[9],DCC[3]PHD3[10]及本身發(fā)生以泛素化和蛋白水解[3,11]。近期實(shí)驗(yàn)研究主要在Ras通路、缺氧反應(yīng)方面研究SIA H2在腫瘤中的作用,一方面已證明SIA H2是RAS通路最下游的因子,抑制SIA H2后,腫瘤生長受到抑制[12-13];另一方面在腫瘤低氧時(shí),SIA H2可以促進(jìn)PHD泛素化降解進(jìn)而使 HIF-1α增多,所以抑制SIA H2就可以抑制 HIF-1α減少,使腫瘤生長受到抑制[5,10]。以上的研究結(jié)果都暗示,SIA H2具有促進(jìn)腫瘤生長的作用,但SIA H2通過什么方式促進(jìn)腫瘤的生長仍是不明的。我們的研究結(jié)果,SIA H2在正常乳腺組織、乳腺導(dǎo)管原位癌、乳腺浸潤性導(dǎo)管癌中陽性率呈遞增趨勢(shì),另外SIA H2在乳腺導(dǎo)管原位癌、乳腺浸潤性導(dǎo)管癌中陽性表達(dá)率明顯高于正常乳腺組織且具有統(tǒng)計(jì)學(xué)意義(P<0.05)。SIA H2蛋白在乳腺癌組織中表達(dá)強(qiáng)于配對(duì)的癌旁正常乳腺組織,二者有明顯的差異,具有統(tǒng)計(jì)學(xué)意義(P<0.05)。在乳腺癌中SIA H2表達(dá)與患者組織學(xué)分級(jí)相關(guān)。這也同Schmidt研究相似,SIA H2在有增殖能力的細(xì)胞中表達(dá)明顯[12]。通過我們的研究,可以推斷SIA H2與乳腺癌發(fā)展有關(guān)。

ERK是MAPK(Mitogen Activated protein kinase,絲裂原活化蛋白激酶)家族成員之一,包括兩個(gè)重要成員ERK1/ERK2。ERK在MAPK途徑中不僅作為轉(zhuǎn)錄因子,而且是膜蛋白[14]。P-ERK是ERK活化的形式。在對(duì)于 ERK的研究中發(fā)現(xiàn),PERK異常或過度表達(dá)在細(xì)胞惡性轉(zhuǎn)化及演進(jìn)中起重要作用[15]。Schmidt研究表明SIA H2是RAS介導(dǎo)的腫瘤發(fā)生所需要的,在胰腺癌細(xì)胞系中抑制SIA H2功能后 ERK/MAPK信號(hào)也減弱[12-13]。在另一項(xiàng)研究中,SIA H2可以引起Sprouty2的降解[16],而Sprouty2可以調(diào)控 P-ERK。以上研究都表明SIA H2可能與P-ERK具有相關(guān)性。我們的統(tǒng)計(jì)學(xué)結(jié)果分析出SIA H2與P-ERK表達(dá)具有正相關(guān)性。在MDA-MB-435S細(xì)胞系轉(zhuǎn)染SIA H2-siRNA后,P-ERK表達(dá)也明顯減少。通過以上結(jié)果,我們可以推斷出SIA H2可能通過 ERK通路發(fā)揮促進(jìn)乳腺腫瘤生長作用。

我們的統(tǒng)計(jì)學(xué)分析結(jié)果表明在 ER和 PgR高表達(dá)的乳腺癌中SIA H2陽性率高。Maurice[17]的研究結(jié)果表明在雌激素受體(ER)陽性乳腺癌細(xì)胞系中,SIA H2的mRNA水平是明顯上調(diào)的,我們的統(tǒng)計(jì)學(xué)分析結(jié)果與其是一致的。但我們?cè)诜治鯯IA H2在乳腺癌細(xì)胞系表達(dá)時(shí)發(fā)現(xiàn),SIA H2在ER低表達(dá)的MDA-MB-435S細(xì)胞系中蛋白表達(dá)強(qiáng)于ER高表達(dá)的MCF-7細(xì)胞系中SIA H2蛋白表達(dá),可能是因?yàn)樵谇忠u能力強(qiáng)的細(xì)胞系中SIA H2蛋白表達(dá)更強(qiáng)。

綜上所述,SIA H2和 P-ERK在乳腺癌中都異常表達(dá),并且二者具有正相關(guān)性。所以我們推斷,SIA H2可能通過調(diào)控ERK途徑在乳腺癌中發(fā)揮重要的作用,通過將SIA H2作為靶點(diǎn)進(jìn)而抑制 ERK通路,可為乳腺癌的治療提供新思路。

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Expression of SIAH2 and P-ERK in breast cancer and its clinical signif icances

Meng Yuan,Dong Jingyu3,Zhen Juan,Sun Jie,Song Min1,Li Bailin2＊
(University,Shenyang 110001,China;2Department of Chemisry,College of Basic Medical Sciences,china Medical University,Shenyang 110001,China;392 K in 7-system-class,China Medical University,Shenyang 110001,China)

Objective To investigate the expression of SIA H2 and P-ERK in human breast cell lines and breast tissues and their correlation in the malignant progression of breast carcinoma.Methods The expression of SIA H2 and P-ERK was examined in 140 paraffin-embeded specimens using immunohistochemical staining.Human breast cells and 23 cases of breast cancer and pairing non-neoplastic tissue were subjected to Western blot for SIA H2 and P-ERK proteins.The expression of P-ERK in SIA H2 siRNA transfected cells was examined by Western blot.Results The expression of SIA H2 and P-ERK was gradually increased in the mabignant progression of breast carcinoma.Western blot analysis showed that the expression of SIA H2 and P-ERK proteins were higher in breast cancer tissues and breast cancer cells than in the adjacent normal breast tissues and normal breast cells.Western blot anaysis also showed that,compared with that of the control cells,the expression of P-ERK in SIA H2 siRNA-transfected cells was significantly decreased.Conclusion The results strongly suggest that SIA H2 and P-ERK are both over expression in human breast cancer.When the expression of SIA H2 was decreased the expression of P-ERK was also decreased.So we conclude that loss or reduction of SIA H2 expression would suppress the expression of PERK in breast cancer cells,which would inhibit tumor cell growth.

Breast carcinoma; SIA H2; P-ERK; Clinicopathological factors; Interference

R737.9

A

10.3870/zgzzhx.2011.03.004

2010-02-10

2011-04-01

遼寧省教育廳科研基金資助項(xiàng)目(2008739)

孟媛,女(1980年),漢族,碩士研究生。

＊通訊作者(To whom correspondence should be addressed)