SIAH1與Bim在乳腺癌中的表達(dá)及意義

1、附件 2論文中英文摘要格式作者姓名： 溫媛媛論文題目 ：SIAH1 與 Bim 在乳腺癌中的表達(dá)及意義作者簡介 ：溫媛媛，女， 1982 年 2 月出生， 2005 年 8 月師從于中國醫(yī)科大學(xué)病理學(xué)教研室宋敏教授，于2010 年 7 月獲博士學(xué)位。中文摘要前言乳腺癌是女性一種常見的惡性腫瘤，已成為女性死亡的主要原因。乳腺癌的發(fā)生經(jīng)歷了以下幾個階段：正常普通導(dǎo)管增生不典型增生導(dǎo)管原位癌乳腺浸潤性導(dǎo)管癌，探討與乳腺癌動態(tài)發(fā)展過程相關(guān)的基因?qū)χ委熑橄侔┚哂兄匾饬x。SIAH 家族蛋白是果蠅屬SINA 蛋白的同系物，人類有兩個高度保守的同源SINA：SIAH1 和SIAH2，siah1 定位于 16

2、q12 號染色體，編碼 282 個氨基酸，與果蠅屬sina 有 76的氨基酸相似， siah2 定位于 3q25 號染色體，編碼了 324 個氨基酸，與果蠅屬 sina 有 68的同一性，與 siah1 有 77的同一性。然而 SIAH1和 SIAH2蛋白在 N端有顯著差別。 SIAH1 蛋白具有 E3 泛素連接酶的活性，可調(diào)節(jié)一些蛋白的泛素化和降解， 這些蛋白包括 ?-catenin ，c-myb，APC和 SIAH1本身。 SIAH1 可作為一種腫瘤抑制因子發(fā)揮生物學(xué)作用，研究發(fā)現(xiàn) SIAH1 在肝癌細(xì)胞系中低表達(dá)且過表達(dá) SIAH1后可誘導(dǎo)肝癌細(xì)胞的凋亡。乳腺癌細(xì)胞過表達(dá) SIAH1后可

3、通過改變有絲分裂來抑制細(xì)胞生長和誘導(dǎo)細(xì)胞凋亡。還有研究表明 SIAH1可以在 p53 依賴和獨(dú)立的細(xì)胞凋亡和腫瘤抑制細(xì)胞模型中表達(dá)。Bim (Bcl-2 interacting mediator of cell death) 成員，是一種重要的凋亡調(diào)節(jié)蛋白?，F(xiàn)已明確，是 Bcl-2 家族中 BH3-only 亞家族的Bim 分子在一定的凋亡刺激下被激活，活化的Bim 分子即可通過與 Bcl-2/Bax的相互作用激活Bax，從而引起線粒體途徑的細(xì)胞凋亡。最近有研究發(fā)現(xiàn) SIAH1 可能通過 JNK/c-jun 途徑來調(diào)節(jié)細(xì)胞凋亡。 JNK信號通路在細(xì)胞應(yīng)激和細(xì)胞凋亡中起重要作用， 且 JNK可通

4、過調(diào)控 Bim 的表達(dá)來誘導(dǎo)細(xì)胞凋亡。 所以我們推測 SIAH1可能通過 JNK 通路來調(diào)控Bim，從而誘導(dǎo)乳腺癌細(xì)胞的凋亡，但上述假設(shè)是否成立？其具體機(jī)制如何？這些問題需要我們進(jìn)一步研究證實。本研究目的旨在探討乳腺組織中SIAH1 和 Bim 的表達(dá)情況及與臨床病理學(xué)參數(shù)的意義。在細(xì)胞水平上，通過過表達(dá)和干擾SIAH1，觀察其對 Bim 表達(dá)， MAPK信號通路活性及對乳腺癌細(xì)1胞凋亡和生長侵襲能力的影響。材料與方法1、乳腺組織本研究選取了 231 例乳腺組織標(biāo)本（包括40 例正常乳腺組織， 53 例乳腺不典型增生組織，36 例乳腺導(dǎo)管原位癌組織和102 例乳腺浸潤性導(dǎo)管癌組織） ，均來自中

5、國醫(yī)科大學(xué)附屬第一臨床學(xué)院 2005-2008 年腫瘤外科手術(shù)切除的標(biāo)本，所有患者術(shù)前均未接受放、化療。標(biāo)本經(jīng)4%中性甲醛固定，石蠟包埋， HE常規(guī)染色后明確診斷。其中部分癌旁乳腺組織（遠(yuǎn)離腫瘤至少5 厘米）和癌組織離體后立即放入液氮后-70 冰箱保存?zhèn)溆谩?、免疫組織化學(xué)染色及結(jié)果判定標(biāo)本用中性福爾馬林溶液固定，石蠟包埋，制成4 m切片，經(jīng)脫蠟，脫苯，水化后，采用鏈菌素抗生物素蛋白 - 過氧化物酶免疫組化法（S-P 法）檢測 SIAH1和 Bim 蛋白表達(dá)。多克隆抗體 SIAH1 和 Bim4孵育過夜。以 PBS緩沖液代替一抗為陰性對照。結(jié)果判定： SIAH1和 Bim 以細(xì)胞質(zhì)中出現(xiàn)棕黃色

6、顆粒為陽性顯色。 高倍視野（× 400）下選陽性信號最強(qiáng)區(qū)域計數(shù) 200 個腫瘤細(xì)胞中陽性細(xì)胞數(shù)，按SIAH1 和 Bim 表達(dá)百分率分為以下四個等級：0%為 0 分，1%-50%為 1分，51%-75%為 2 分， >75%為 3 分。根據(jù)免疫組化染色強(qiáng)度分為三個等級：淺黃色計為1 分，棕黃色計為 2 分，黃褐色計為3 分。以陽性細(xì)胞率和染色強(qiáng)度的分值乘積作為每一例的積分，積分<4 者判定為陰性，積分 4 為陽性。3、細(xì)胞培養(yǎng)用含 10%新鮮胎牛血清的DMEM培養(yǎng)基，在37， 5%CO2的條件下培養(yǎng)人乳腺癌細(xì)胞系MDA-MB-231和 MCF7，人乳腺正常上皮細(xì)胞系MC

7、F7-10A。4、Western Blot在收集的細(xì)胞內(nèi)加入裂解液充分裂解，低溫高速離心（4， 12000 轉(zhuǎn) /min ， 30min），提取上清為總蛋白。上樣蛋白量為 60g。電泳（ 12%SDS-PAGE凝膠）、轉(zhuǎn)印、封閉，一抗 SIAH1（1:400 ）、Bim（1:1000 ）、JNK（ 1:1000 ）、p-JNK（1:1000 ）、ERK（1:400 ）、p-ERK（ 1:400 ）、p38（1:400 ）、p-p38（1:400 ）和 -actin （1:200 ），4孵育過夜，分別與各自對應(yīng)的二抗（1:4000 ）室溫孵育 2 小時， ECL顯色，結(jié)果經(jīng)自動凝膠成像分析儀采集

8、，進(jìn)行灰度值測定。5、RT-PCR采用 TrizolTM 試劑提取乳腺癌組織或培養(yǎng)細(xì)胞的總RNA，利用 RNAPCRKit (AMV) Ver. 3.0試劑盒進(jìn)行反轉(zhuǎn)錄。分別擴(kuò)增SIAH1 和 Bim，以 -actin為內(nèi)參。擴(kuò)增產(chǎn)物經(jīng)1.5%瓊脂糖凝膠電泳后，成像分析。6、siRNA干擾2根據(jù) siRNA 設(shè)計原則，選取人 SIAH1 mRNA和 Bim mRNA中的特異性核苷酸片段為靶目標(biāo)，應(yīng)用 Ambion 公司在線 siRNA設(shè)計軟件設(shè)計 SIAH1 和 Bim 的 RNAi序列，經(jīng) Blast 確定所選靶向的基因是特異的，序列由上海吉凱基因化學(xué)技術(shù)有限公司合成。實驗分三組：空白對照組

9、、非特異性 siRNA轉(zhuǎn)染組和特異性siRNA 轉(zhuǎn)染組，每個實驗均重復(fù)三次，轉(zhuǎn)染具體步驟按Lipofect2000 試劑說明書進(jìn)行。 SIAH1siRNA 序列： 1： 5'-AACTCCTGCCTCCTTATGTATTT，-3'2 ： 5'-GAUAGGAACACGCAAGCAA，-3'：5'-GUUGCAUGUAGUAACACUA。-3'非 特 異 性siRNA1 （ controlsiRNA1 ） 序 列 ：5'-AAGAGCCGTCAGACTGCTACA。-Bim3' siRNA 序 列 ： 1 ： 5'-ACCG

10、AGAAGGUAGACAAUU，-23'：5'-CUACCUCCCUACAGACAGA，-3'：5'-CUACCUCCCUACAGACAGA。非-3'特異性 siRNA 2（controlsiRNA 2）序列： 5'-CGUACGCGGAAUACUUCGA。鑒-3'于轉(zhuǎn)染效率和穩(wěn)定性，我們選擇SIAH1 siRNA1 和 Bim siRNA 1 進(jìn)行實驗。7、細(xì)胞轉(zhuǎn)染SIAH1表達(dá)質(zhì)粒 pcDNA3-myc-SIAH1和對照質(zhì)粒 pcDNA3-myc由 Matsuzawa Shu-ichi （BurnhamInstitute，La Jol

11、la ， California，USA）教授惠贈。具體轉(zhuǎn)染步驟按脂質(zhì)體LipofectamineTM試劑說明書進(jìn)行。以未轉(zhuǎn)染的細(xì)胞和轉(zhuǎn)染空載體細(xì)胞作為對照。8、MTT法檢測細(xì)胞增殖將單個細(xì)胞懸液接種于96 孔培養(yǎng)板中，每孔含104 個細(xì)胞，培養(yǎng)24 小時，每孔加入MTT（Methylthiazolyldiphenyl-tetrazoliumbromide ）溶液繼續(xù)培養(yǎng)4 小時，加入 DMSO，490nm波長下測定各孔吸收值，以不含細(xì)胞的等體積培養(yǎng)基作對照。繪制細(xì)胞生長曲線。9、細(xì)胞侵襲能力檢測在 transwell 小室下室加入 600l 含 10%胎牛血清 DMEM培養(yǎng)基，上室加入 100

12、 l 預(yù)冷的用無血清 DMEM培養(yǎng)基稀釋的 Matrigel （1:7 ），將轉(zhuǎn)染后 24 小時的細(xì)胞接種到上室。 37，5%CO2孵箱中培養(yǎng) 32 小時后吸盡培養(yǎng)基， PBS清洗后，甲醇室溫固定15 分鐘，用棉簽擦掉微孔膜上表面的細(xì)胞，蘇木素染色，室溫干燥過夜。取下微孔膜，置載玻片上，鏡下觀察。10、流式細(xì)胞儀檢測細(xì)胞凋亡收集對數(shù)生長期細(xì)胞制成 1× 107 個/ml 細(xì)胞懸液。取 1ml 細(xì)胞懸液 75%冷乙醇于 4固定、離心后制成 500 l 的細(xì)胞懸液，加碘化丙啶染液于 4孵育 45 分鐘后上機(jī)檢測， ModFitLT3.0 軟件分析細(xì)胞凋亡。 sub-G1 期細(xì)胞代表凋亡細(xì)

13、胞。11、統(tǒng)計分析各組資料利用 SPSS for Window 13.0進(jìn)行統(tǒng)計分析。 P <0.05 有統(tǒng)計學(xué)意義。結(jié)果31、SIAH1在乳腺癌中低表達(dá)，且可通過上調(diào)Bim 表達(dá)來誘導(dǎo)乳腺癌細(xì)胞凋亡（1）SIAH1 和 Bim 蛋白在乳腺癌癌變過程中表達(dá)逐漸下調(diào)，并且二者表達(dá)與乳腺癌分期和分化相關(guān)：免疫組化結(jié)果顯示，SIAH1 和 Bim 蛋白在乳腺正常組織，乳腺不典型增生，乳腺導(dǎo)管原位癌和乳腺浸潤性導(dǎo)管癌中的陽性表達(dá)率分別為85.0 與 87.5 ，66.0 與 67.9 ，52.8與 63.9 ， 28.4 and 34.3 。統(tǒng)計學(xué)結(jié)果顯示：SIAH1（P=0.002）和 Bim

14、（P=0.008）蛋白在乳腺正常組織和導(dǎo)管原位癌中表達(dá)有明顯差別，SIAH1（P=0.016）和 Bim（P=0.002）蛋白在導(dǎo)管原位癌和浸潤性導(dǎo)管癌中表達(dá)也有明顯統(tǒng)計學(xué)意義。在 231 例乳腺組織中， SIAH1 與 Bim蛋白表達(dá)呈正相關(guān)（ P < 0.001 ，rs=0.395 ）。 SIAH1（ P=0.025 和 P=0.018）與 Bim（ P=0.045和 P=0.032）蛋白在高分化和早期乳腺癌中呈高表達(dá)。（2）SIAH1 mRNA和蛋白在乳腺癌組織和乳腺癌細(xì)胞系中低表達(dá)：RT-PCR和 Western blot結(jié)果顯示， SIAH1 mRNA（ P<0.05）與

15、 蛋白（ P<0.05）在乳腺癌組織表達(dá)低于癌旁乳腺組織，在乳腺癌細(xì)胞系 MDA-MB-231和 MCF-7中表達(dá)低于乳腺正常上皮細(xì)胞系 MCF-10A（ P<0.05）。（3）過表達(dá) SIAH1后可上調(diào) Bim 的表達(dá)，從而誘導(dǎo)乳腺癌細(xì)胞凋亡：實驗結(jié)果表明，與乳腺癌細(xì)胞系 MDA-MB-231和 MCF-7轉(zhuǎn)染空質(zhì)粒組相比， 轉(zhuǎn)染 pcDNA3-myc-SIAH1后，Bim（P<0.05）表達(dá)上調(diào)，細(xì)胞凋亡率（ P<0.05）增加；與乳腺癌細(xì)胞系 MDA-MB-231和 MCF-7 轉(zhuǎn)染pcDNA3-myc-SIAH1相比，共轉(zhuǎn)染 pcDNA3-myc-SIAH1和

16、SIAH1 siRNA 1 后，凋亡率（ P<0.05）明顯減少。（ 4）過表達(dá) SIAH1以不依賴 Bim 的途徑來抑制乳腺癌細(xì)胞的侵襲： 細(xì)胞侵襲實驗表明， 與乳腺癌細(xì)胞系 MDA-MB-231和 MCF-7轉(zhuǎn)染 control siRNA 1 組（ 13.26 ±2.18 與 11.43 ±1.83 ）或未轉(zhuǎn)染組（ 15.45 ±2.36 與 11.69 ±1.62 ）相比，轉(zhuǎn)染 SIAH1 siRNA 1 組（ 55.21 ±5.23 與43.17 ±4.12 ）后，乳腺癌細(xì)胞侵襲力（P0.05 ）增加。相反的，乳腺癌細(xì)

17、胞系MDA-MB-231和 MCF-7轉(zhuǎn)染 pcDNA3-myc-SIAH1（3.21 ± 0.96 與 2.93 ±0.73 ）后，細(xì)胞侵襲力比轉(zhuǎn)染pcDNA3-myc組（ 14.36 ±2.32 與 11.73 ±1.76 ）或未轉(zhuǎn)染組（ 15.45 ±2.36 與 11.69 ± 1.62 ）降低。乳腺癌細(xì)胞系 MDA-MB-231和 MCF-7共轉(zhuǎn)染 pcDNA3-myc-SIAH1與 Bim siRNA 1 （3.06 ±0.89 與 2.09 ±0.68 ）后，細(xì)胞侵襲力與單獨(dú)轉(zhuǎn)染pcDNA3-myc-

18、SIAH1組（ 3.21 ±0.96 與 2.93 ± 0.73 ）相比無明顯差別。2、 SIAH1 可激活 JNK通路上調(diào) Bim 表達(dá)來誘導(dǎo)乳腺癌細(xì)胞凋亡， 且可使 ERK通路失活來抑制乳腺癌細(xì)胞侵襲（1）SIAH1 在乳腺癌細(xì)胞系中可調(diào)控 MAPK通路的活性： Western blot 結(jié)果顯示，過表達(dá) SIAH1 后可上調(diào) P-JNK 蛋白的表達(dá)且可下調(diào) P-ERK蛋白的表達(dá)。相反的，干擾 SIAH1后可下調(diào) P-JNK 蛋白的表達(dá)且可上調(diào) P-ERK蛋白的表達(dá)。過表達(dá)或干擾 SIAH1 對 P-P38 蛋白的表達(dá)無明4顯影響。（2）過表達(dá) SIAH1 可通過激活

19、JNK/Bim 通路來誘導(dǎo)乳腺癌細(xì)胞凋亡： Western blot 結(jié)果表明，采用 JNK通路的特異性抑制劑 SP600125后，過表達(dá) SIAH1導(dǎo)致的 P-JNK和 Bim 蛋白表達(dá)上調(diào)被抑制。流式結(jié)果顯示，過表達(dá) SIAH1誘導(dǎo)的乳腺癌細(xì)胞凋亡可被 SP600125抑制。（3）過表達(dá) SIAH1可通過 ERK通路來抑制乳腺癌細(xì)胞侵襲：Western blot結(jié)果表明，采用 ERK通路的特異性抑制劑PD98059 后，干擾SIAH1 導(dǎo)致的P-ERK 蛋白表達(dá)上調(diào)被抑制。Transwell結(jié)果表明，干擾SIAH1導(dǎo)致的乳腺癌細(xì)胞侵襲力增加可被PD98059抑制。（4）JNK 通路和 ER

20、K通路同時影響 SIAH1 調(diào)節(jié)的乳腺癌細(xì)胞生長： MTT結(jié)果顯示，過表達(dá)SIAH1抑制乳腺癌細(xì)胞的生長可被 SP600125影響。同時，我們觀察到干擾 SIAH1 促進(jìn)乳腺癌細(xì)胞的生長可被 PD98059抑制。結(jié)論1、SIAH1 在乳腺癌癌變過程中可作為一種腫瘤抑制因子發(fā)揮作用，SIAH1 與 Bim 表達(dá)呈正相關(guān)，并且它們均與乳腺癌的臨床病理分期和分化密切相關(guān)。2、 在乳腺癌細(xì)胞中SIAH1 通過調(diào)控 JNK通路的活性來上調(diào)Bim 表達(dá)，促進(jìn)乳腺癌細(xì)胞凋亡。3、 在乳腺癌細(xì)胞中SIAH1 通過調(diào)控 ERK通路的活性來抑制乳腺癌細(xì)胞侵襲，且不依賴于Bim 的表達(dá)。關(guān)鍵詞： 乳腺癌； SIAH

21、1；Bim；JNK；ERK；凋亡；侵襲The expression and significance of SIAH1 and Bim in breast cancerWen YuanyuanABSTRACTIntroductionBreast cancer is the most leading cause of cancer death in the women all over the world. The occurrence of breast cancer experienced several stages: normal, normal ductal hyperplasis,

22、atypical ductal hyperplasis, ductal carcinomain situ and invasive ductal carcinoma. Thus, acquire of new target molecules that play important roles in breast carcinogenesis will be essential for improving therapeutic intervention and prognosis of breast cancers.SIAH (seven in absentia homolog) prote

23、ins are homologues of Drosophila seven in absentia5(SINA) protein. SINA has two human homologues, SIAH1 and SIAH2. Siah1 locates in chromosome 16q12 and encodes a 282-amino-acid protein with 76% amino acid identity to the Drosophila SINA protein. Siah2 maps to chromosome 3q25 and encodes a 324-amino

24、-acid protein that shares 68%identity with Drosophila SINA and 77% identity with human SIAH1. SIAH1 and SIAH2 protein differ significantly at their N termini. SIAH1 has been described to have E3 ubiquitin ligase activity and target some proteins, such as ?-catenin, c-myb, APC and SIAH1, for ubiquiti

25、n mediate degradation. SIAH1 may function as a tumor suppressor gene. The study showed that SIAH1expression was lower in HCC cell lines, and overexpression of SIAH1 could induce apoptosis of HCC cells. Some reports showed that SIAH1 overexpression inhibited cell growth and increased apoptosisthrough

26、 major alteration of mitosis. Other showed that SIAH1 was activated during apoptosis and tumor suppression in p53-dependent andindependent cellular models.The BH3-only protein Bim (Bcl-2-interacting mediator of cell death) is a member of Bcl-2 famliy.It is a critical mediator of cell apoptosis in va

27、rious cell types. It was reported that the activation of Bim by certain sitmulation of apoptosis was interacted with Bcl-2/Bax, inducing mitochondrial pathway of apoptosis.The emerging evidence showed that SIAH1 could induce apoptosis by activating the c-Jun NH2-terminal kinade (JNK) pathway. And JN

28、K pathway played an important role in cell apoptosis. Some studies suggested that Bim was involved in JNK-dependent apoptosis. So we hypothesized that SIAH1 might induce apoptosis by up-regulating the expression of Bim via JNK pathway. It is necessary to confirm this suppose in our further studies.I

29、n our present study, we implored the expression of SIAH1 and Bim in breast tissues and analyzedthe relationship between SIAH1 expression and clinicalpathology parameters. In addition, weexamined the overexpression or knockdown of SIAH1 had an effect on the expression of Bim, theactivity of MAPK path

30、way, apoptosis and invasion in breast cancer cells.Materials and Methods1. Patients and specimensA total of 231 cases of breast tissues (including 40 cases of normal breast tissues (NBT), 53 cases of atypical ductal hyperplasia (ADH), 36 cases of ductal carcinoma in situ (DCIS), and 102 cases of inv

31、asive ductal carcinoma (IDC)were obtained from the January 1st, 2005 to the December31st, 2008 at the First Affiliated Hospital of China Medical University. All of the enrolled patients underwentcurative surgical resection withouthaving chemotherapy or radiationtherapy. Formalin-fixed6paraffin-embed

32、ded sections of tissues obtained from surgical samples were stained routinely with hematoxylin and eosin (H&E). Among these samples, some fresh specimens and corresponding normal tissue samples were stored at -70C immediately° after resection until the extraction of protein and RNA.2. Immun

33、ohistochemical study and result assessmentFour-micron thick sections were prepared from the paraffin-embedded tissues.Immunostaining was performed by the streptavidin-peroxidase (S-P) method. For negative control, the primary antibodies were replaced by non-immune serum. Brown particles appearing in

34、 cytoplasm was as regarded as positive cells. The intensity of immunostaining (1=weak, 2=moderate, and 3=intense)and the percentage of positive cells (0%=negative, 1-50%=1, 5175%=2,- 76%=3) were assessed in atleast 5 high power fields (400 magnification)×. The scores of each sample were multipl

35、ied to give afinal score of 0, 1, 2, 3, 4, 6 or 9, and the tissues were finally determined as negative if score < 4; andpositive expression if score 4.3. Cell cultureHuman breast cancer cell lines MCF-7, MDA-MB-231, and human breast epithelial MCF-10A cell line were maintained in Dulbcco's Mo

36、difed Eagle Medium (DMEM) supplemented 10% fetal bovine serum in a humidified atmosphere with 5% of CO2.4. Western blotThe protein was extracted with lysis buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 2 g/ml aprotinin, 1 mM PMSF) for 1 hour at 4 C. The°supernatants were centrifuged at 12000 rpm for

37、 30 minutes at 4 ° C. The supernatants containing total protein were harvested. Aliquots containing 60 proteins were separated on a 12% SDS-polyacrylamide gel and transferred to Polyvinylidene Fluoride(PVDF) membranes. After blocking, the blots were respectively incubated with primary antibody

38、directed against SIAH1 (1:400), Bim (1:1000), JNK (1:1000), p-JNK (1:1000), ERK (1:400), p-ERK (1:400), p38 (1:400), p-p38 (1:400) or -actin (1:200) overnight at 4°C and followed by eachcorresponding second antibody at room temperature for 2 hour at 37C. Then the results° developed by ECL.

39、 The protein bands were then analyzed using the Bioimaging System. The grayscale values of the SIAH1 and Bim bands were normalized to the values of the corresponding -actin band todetermine the expression of the protein.5. RT-PCRTotal RNA was isolated using Trizol Reagent according to the manufactur

40、er s instruct7 s protocol-actin. served as an internal control. After electrophoresis, the PCR productswere stained withethidium bromide and analyzed using a Bioimaging system. Relative bandintensities were determined using NIH image software.6. Small RNA InterferenceThe small interfering RNA (siRNA

41、) duplexes were synthesized and purified by Ji Kai. SIAH1targetsequencewasasfollows:1:5'-AACTCCTGCCTCCTTATGTATTT-3'2:5'-GAUAGGAACACGCAAGCAA-3'3: 5'-GUUGCAUGUAGUAACACUA-3'. The nonsilencingsiRNA 1 (control siRNA 1) sequence was as follows: 5'-AAGAGCCGTCAGACTGCTACA-3'.

42、Bimtargetsequencewasasfollows:1:5'-ACCGAGAAGGUAGACAAUU-3'2:5'-CUACCUCCCUACAGACAGA-3'3:5'-CUACCUCCCUACAGACAGA-3'.ThenonsilencingsiRNA2 (controlsiRNA2) sequence was as follows:5'-CGUACGCGGAAUACUUCGA-3'.Considering the relative effectiveness and stability, SIAH1 siRNA 1

43、and Bim siRNA 1 were selected by comparing our pilot experiments.7. Cell transfectionThe SIAH1 expression vector pcDNA3-myc-SIAH1 and the empty vector pcDNA3-myc were kindly provided by Dr. Matsuzawa Shu-ichi (Burnham Institute, La Jolla, California, USA). The cells were transfected with the SIAH1/B

44、im siRNA or pcDNA3-myc-SIAH1 using Lipofectamine 2000,following the manufacturer's instructions. The control siRNA and pcDNA3-myc were used as negative controls.8. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT) AssayThe transfected cells were seeded in 96-well plates (1 

45、15;104 cells/well). Cell proliferation was evaluated each day for 4 days after transfection using MTT method. The absorbance, which was directly proportional to the mumber of living cells in culture, was measured at 490 nm using amicroplate reader. A blank with dimethyl sulfoxide (DMSO) alone was me

46、asured and subtracted from all values. All the assays were done at least 3 times independently. The cell growth curve was analyzed.9. Matrigel invasion assayCell invasive ability was examined using a 24-well Transwell with 8 mpore polycarbonate memberance inserts according to the manufacturer protoc

47、ols. The Matrigel (100 g/ml1:7) which was precooled with serum-free DMEM was applied to the upper surface of the membranes. After8transfection for 24 hours, cells were seeded on the upper chamber (5104 cells/well) and incubated× for 32 hours. Nontransfected cells served as the control. Cells th

48、at had invaded the surface of the membrane were fixed with methanol and stained with hematoxylin. Five random high-magnification microscope fields per filter were counted.10. Flow cytometry (FCM)After 48 hours of culture, cells from each experimental group were collected and digested with trypsin an

49、d fixed with 75% ice-cold ethanol at 4 C overnight. Cells° (1 107) ×were centrifuged at 1000 rpm 5 minutes, and were resuspendedwith 50 g/mlpropidium iodide for 45 minutes in the dark before analysis. The percentages of cells in the different cell cycle phases were determined using a FACSC

50、alibur Flow Cytometer with CellQuest 3.0 software.11. Statistical analysisAll statistical calculations were performed by SPSS 13.0 for Windows software. P values less than 0.05 were considered statistically significant.Results1、SIAH1 was down-regulated in breast cancer tissues and cell, and inducing

51、 apoptosis of breast cancer cells by up-regulating the level of Bim（1）The expression of SIAH1 and Bim significantly decreased concurrently in the process of breast carcinogenesis and were correlated with well to moderately-differentiated and early-stage breast cancer. The immunohistochemistry result

52、s showed there was a decreasing tendency in positive rate of SIAH1 and Bim expression from normal breast tissues (NBT) (85.0and 87.5, respectively), atypical ductal hyperplasia (ADH) (66.0 and 67.9, respectively), ductal carcinoma in situ (DCIS)(52.8 and 63.9 , respectively) to invasive ductal carci

53、noma (IDC) (28.4and 34.3, respectively).There were statistically significant difference of SIAH1 (P=0.002 and 0.008, respectively) and Bim(P= 0.016 and 0.002, respectively) expression between the NBT and DCIS and between the DCIS andIDC. The Spearman s correlation test revealed that the SIAH1 expres

54、sion was significantly positivelyassociated withBimexpression inthe 231 breast specimens (P <0.001, rs=0.395). Theclinicopathological data analysis showed that the expression of SIAH1 (P=0.025) and Bim (P=0.045)weresignificantlylowerinpoorly-differentiatedtumors(gradeIII)thaninwelltomoderately-di

55、fferentiated tumors (grade Iand II).The SIAH1(P=0.018) and Bim(P=0.032)expression were significantlylower in advanced-stage tumors (stage IIIand IV)compared toearly-stage tumors (stage I and II).9（2）SIAH1 mRNA and protein expression were significantly lower in breast cancer tissues and cell linesThe

56、 RT-PCR and Western blot results showed that SIAH1 mRNA (P<0.05) and protein (P<0.05) expression were significantly lower in breast tumor tissues in comparison with the non-tumorous counterparts, and in human breast cancer MDA-MB-231, MCF-7 cell lines in comparison with human normal breast epi

57、thelial MCF-10A cell line (P<0.05).（3）Overexpression of SIAH1 inducing apoptosis of breast cancer cells by up-regulating the level of Bim: Our study showed a moderate increase in apoptosis after MDA-MB-231 and MCF-7 cells transfected with pcDNA3-myc-SIAH1 (approximately 18.06%±3.35% and 19.27%±4.32%, respectively, P 0.05), compared with the cells transfected with the empty vector (approximately 1.11%±0.21% and 0.05%±0.008%, respe