SIAH1与Bim在乳腺癌中的表达及意义
摘要
前言
乳腺癌是女性一种常见的恶性肿瘤,已成为女性死亡的主要原因。乳腺癌的发生经历了以下几个阶段：正常—普通导管增生—不典型增生—导管原位癌—乳腺浸润性导管癌,探讨与乳腺癌动态发展过程相关的基因对治疗乳腺癌具有重要意义。
SIAH家族蛋白是果蝇属SINA蛋白的同系物,人类有两个高度保守的同源SINA:SIAH1和SIAH2, siahl定位于16q12号染色体,编码282个氨基酸,与果蝇属sina有76%的氨基酸相似,siah2定位于3q25号染色体,编码了324个氨基酸,与果蝇属sina有68%的同一性,与siahl有77%的同一性。然而SIAH1和SIAH2蛋白在N端有显著差别。SIAH1蛋白具有E3泛素连接酶的活性,可调节一些蛋白的泛素化和降解,这些蛋白包括B-catenin, c-myb, APC和SIAH1本身。SIAH1可作为一种肿瘤抑制因子发挥生物学作用,研究发现SIAH1在肝癌细胞系中低表达且过表达SIAH1后可诱导肝癌细胞的凋亡。乳腺癌细胞过表达SIAH1后可通过改变有丝分裂来抑制细胞生长和诱导细胞凋亡。还有研究表明SIAH1可以在p53依赖和独立的细胞凋亡和肿瘤抑制细胞模型中表达。
Bim (Bcl-2 interacting mediator of cell death)是Bcl-2家族中BH3-only亚家族的成员,是一种重要的凋亡调节蛋白。现已明确,Bim分子在一定的凋亡刺激下被激活,活化的Bim分子即可通过与Bcl-2/Bax的相互作用激活Bax,从而引起线粒体途径的细胞凋亡。
最近有研究发现SIAH1可能通过JNK/c-jun途径来调节细胞凋亡。JNK信号通路在细胞应激和细胞凋亡中起重要作用,且JNK可通过调控Bim的表达来诱导细胞凋亡。所以我们推测SIAH1可能通过JNK通路来调控Bim,从而诱导乳腺癌细胞的凋亡,但上述假设是否成立?其具体机制如何?这些问题需要我们进一步研究证实。
本研究目的旨在探讨乳腺组织中SIAH1和Bim的表达情况及与临床病理学参数的意义。在细胞水平上,通过过表达和干扰SIAH1,观察其对Bim表达,MAPK信号通路活性及对乳腺癌细胞凋亡和生长侵袭能力的影响。
材料与方法
1、乳腺组织
本研究选取了231例乳腺组织标本(包括40例正常乳腺组织,53例乳腺不典型增生组织,36例乳腺导管原位癌组织和102例乳腺浸润性导管癌组织),均来自中国医科大学附属第一临床学院2005-2008年肿瘤外科手术切除的标本,所有患者术前均未接受放、化疗。标本经4%中性甲醛固定,石蜡包埋,HE常规染色后明确诊断。其中部分癌旁乳腺组织(远离肿瘤至少5厘米)和癌组织离体后立即放入液氮后-70℃冰箱保存备用。
2、免疫组织化学染色及结果判定
标本用中性福尔马林溶液固定,石蜡包埋,制成4μm切片,经脱蜡,脱苯,水化后,采用链菌素抗生物素蛋白-过氧化物酶免疫组化法(S-P法)检测SIAH1和Bim蛋白表达。多克隆抗体SIAH1和Bim4℃孵育过夜。以PBS缓冲液代替抗为阴性对照。结果判定：SIAH1和Bim以细胞质中出现棕黄色颗粒为阳性显色。高倍视野(×400)下选阳性信号最强区域计数200个肿瘤细胞中阳性细胞数,按SIAH1和Bim表达百分率分为以下四个等级：0%为0分,1%-50%为1分,51%-75%为2分,>75%为3分。根据免疫组化染色强度分为三个等级：浅黄色计为1分,棕黄色计为2分,黄褐色计为3分。以阳性细胞率和染色强度的分值乘积作为每一例的积分,积分<4者判定为阴性,积分≥4为阳性。
3、细胞培养
用含10%新鲜胎牛血清的DMEM培养基,在37℃,5%CO2的条件下培养人乳腺癌细胞系MDA-MB-231和MCF7,人乳腺正常上皮细胞系MCF7-10A。
4、Western Blot
在收集的细胞内加入裂解液充分裂解,低温高速离心(4℃,12000转／min,30min),提取上清为总蛋白。上样蛋白量为60μg。电泳(12%SDS-PAGE凝胶)、转印、封闭,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β-actin (1:200),4℃孵育过夜,分别与各自对应的二抗(1：4000)室温孵育2小时,ECL显色,结果经自动凝胶成像分析仪采集,进行灰度值测定。
5、RT-PCR
采用TrizolTM试剂提取乳腺癌组织或培养细胞的总RNA,利用RNA PCR Kit (AMV)Ver.3.0试剂盒进行反转录。分别扩增SIAH1和Bim,以β-actin为内参。扩增产物经1.5%琼脂糖凝胶电泳后,成像分析。
6、siRNA干扰
根据siRNA设计原则,选取人SIAH1 mRNA和Bim mRNA中的特异性核苷酸片段为靶目标,应用Ambion公司在线siRNA设计软件设计SIAH1和Bim的RNAi序列,经Blast确定所选靶向的基因是特异的,序列由上海吉凯基因化学技术有限公司合成。实验分三组：空白对照组、非特异性siRNA转染组和特异性siRNA转染组,每个实验均重复三次,转染具体步骤按Lipofect 2000试剂说明书进行。SIAH1 siRNA序列：1:5'-AACTCCTGCCTCCTTATGTATTT-3',2:5'-GAUAGGAAC ACGCAAGCAA-3',3:5'-GUUGCAUGUAGUAACACUA-3'。非特异性siRNA 1 (control siRNA 1)序列：5'-AAGAGCCGTCAGACTGCTACA-3'。Bim siRNA序列：1:5'-ACCGAGAAGGUAGACAAUU-3',2:5'-CUACCUCCCUACAGACA GA-3',3:5'-CUACCUCCCUACAGACAGA-3'。非特异性siRNA 2 (control siRNA 2)序列：5'-CGUACGCGGAAUACUUCGA-3'。鉴于转染效率和稳定性,我们选择SIAH1 siRNA 1和Bim siRNA 1进行实验。
7、细胞转染
SIAH1表达质粒pcDNA3-myc-SIAH1和对照质粒pcDNA3-myc由Matsuzawa Shu-ichi (Burnham Institute, La Jolla, California, USA)教授惠赠。具体转染步骤按脂质体LipofectamineTM试剂说明书进行。以未转染的细胞和转染空载体细胞作为对照。
8、MTT法检测细胞增殖
将单个细胞悬液接种于96孔培养板中,每孔含104个细胞,培养24小时,每孔加入MTT (Methylthiazolyldiphenyl-tetrazolium bromide)溶液继续培养4小时,加入DMSO,490nm波长下测定各孔吸收值,以不含细胞的等体积培养基作对照。绘制细胞生长曲线。
9、细胞侵袭能力检测
在transwell小室下室加入600μl含10%胎牛血清DMEM培养基,上室加入100μl预冷的用无血清DMEM培养基稀释的Matrigel (1:7),将转染后24小时的细胞接种到上室。37℃,5%CO2孵箱中培养32小时后吸尽培养基,PBS清洗后,甲醇室温固定15分钟,用棉签擦掉微孔膜上表面的细胞,苏木素染色,室温干燥过夜。取下微孔膜,置载玻片上,镜下观察。
10、流式细胞仪检测细胞凋亡
收集对数生长期细胞制成1×107个／ml细胞悬液。取1ml细胞悬液75%冷乙醇于4℃固定、离心后制成500μl的细胞悬液,加碘化丙啶染液于4℃孵育45分钟后上机检测,ModFitLT3.0软件分析细胞凋亡。sub-G1期细胞代表凋亡细胞。
11、统计分析
各组资料利用SPSS for Window 13.0进行统计分析。P<0.05有统计学意义。
结果
1、SIAH1在乳腺癌中低表达,且可通过上调Bim表达来诱导乳腺癌细胞凋亡
(1) SIAH1和Bim蛋白在乳腺癌癌变过程中表达逐渐下调,并且二者表达与乳腺癌分期和分化相关：免疫组化结果显示,SIAH1和Bim蛋白在乳腺正常组织,乳腺不典型增生,乳腺导管原位癌和乳腺浸润性导管癌中的阳性表达率分别为85.0%与87.5%,66.0%与67.9%,52.8%与63.9%,28.4%and 34.3%。统计学结果显示：SIAH1 (P=0.002)和Bim (P=0.008)蛋白在乳腺正常组织和导管原位癌中表达有明显差别,SIAH1 (P=0.016)和Bim (P=0.002)蛋白在导管原位癌和浸润性导管癌中表达也有明显统计学意义。在231例乳腺组织中,SIAH1与Bim蛋白表达呈正相关(P<0.001,rs=0.395)。SIAH1 (P=0.025和P=0.018)与Bim(P=0.045和P=0.032)蛋白在高分化和早期乳腺癌中呈高表达。
(2) SIAH1 mRNA和蛋白在乳腺癌组织和乳腺癌细胞系中低表达：RT-PCR和Western blot结果显示,SIAH1 mRNA (P<0.05)与蛋白(P＜0.05)在乳腺癌组织表达低于癌旁乳腺组织,在乳腺癌细胞系MDA-MB-231和MCF-7中表达低于乳腺正常上皮细胞系MCF-10A (P<0.05)。
(3)过表达SIAH1后可上调Bim的表达,从而诱导乳腺癌细胞凋亡：实验结果表明,与乳腺癌细胞系MDA-MB-231和MCF-7转染空质粒组相比,转染pcDNA3-myc-SIAH1后,Bim(P＜0.05)表达上调,细胞凋亡率(P＜0.05)增加;与乳腺癌细胞系MDA-MB-231和MCF-7转染pcDNA3-myc-SIAH1相比,共转染pcDNA3-myc-SIAH1和SIAH1 siRNA 1后,凋亡率(P＜0.05)明显减少。
(4)过表达SIAH1以不依赖Bim的途径来抑制乳腺癌细胞的侵袭：细胞侵袭实验表明,与乳腺癌细胞系MDA-MB-231和MCF-7转染control siRNA 1组(13.26±2.18与11.43±1.83)或未转染组(15.45±2.36与11.69±1.62)相比,转染SIAH1 siRNA 1组(55.21±5.23与43.17±4.12)后,乳腺癌细胞侵袭力(P＜0.05)增加。相反的,乳腺癌细胞系MDA-MB-231和MCF-7转染pcDNA3-myc-SIAH1 (3.21±0.96与2.93±0.73)后,细胞侵袭力比转染pcDNA3-myc组(14.36±2.32与11.73±1.76)或未转染组(15.45±2.36与11.69±1.62)降低。乳腺癌细胞系MDA-MB-231和MCF-7共转染pcDNA3-myc-SIAH1与Bim siRNA 1 (3.06±0.89与2.09±0.68)后,细胞侵袭力与单独转染pcDNA3-myc-SIAH1组(3.21±0.96与2.93±0.73)相比无明显差别。
2、SIAH1可激活JNK通路上调Bim表达来诱导乳腺癌细胞凋亡,且可使ERK通路失活来抑制乳腺癌细胞侵袭
(1) SIAH1在乳腺癌细胞系中可调控MAPK通路的活性：Western blot结果显示,过表达SIAH1后可上调P-JNK蛋白的表达且可下调P-ERK蛋白的表达。相反的,干扰SIAH1后可下调P-JNK蛋白的表达且可上调P-ERK蛋白的表达。过表达或干扰SIAH1对P-P38蛋白的表达无明显影响。
(2)过表达SIAH1可通过激活JNK/Bim通路来诱导乳腺癌细胞凋亡：Western blot结果表明,采用JNK通路的特异性抑制剂SP600125后,过表达SIAH1导致的P-JNK和Bim蛋白表达上调被抑制。流式结果显示,过表达SIAH1诱导的乳腺癌细胞凋亡可被SP600125抑制。
(3)过表达SIAH1可通过ERK通路来抑制乳腺癌细胞侵袭：Western blot结果表明,采用ERK通路的特异性抑制剂PD98059后,干扰SIAH1导致的P-ERK蛋白表达上调被抑制。Transwell结果表明,干扰SIAH1导致的乳腺癌细胞侵袭力增加可被PD98059抑制。
(4)JNK通路和ERK通路同时影响SIAH1调节的乳腺癌细胞生长：MTT结果显示,过表达SIAH1抑制乳腺癌细胞的生长可被SP600125影响。同时,我们观察到干扰SIAH1促进乳腺癌细胞的生长可被PD98059抑制。
结论
1、SIAH1在乳腺癌癌变过程中可作为一种肿瘤抑制因子发挥作用,SIAH1与Bim表达呈正相关,并且它们均与乳腺癌的临床病理分期和分化密切相关。
2、在乳腺癌细胞中SIAH1通过调控JNK通路的活性来上调Bim表达,促进乳腺癌细胞凋亡。
3、在乳腺癌细胞中SIAH1通过调控ERK通路的活性来抑制乳腺癌细胞侵袭,且不依赖于Bim的表达。
Breast 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, 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) proteins are homologues of Drosophila seven in absentia (SINA) protein. SINA has two human homologues, SIAH1 and SIAH2. Siahl 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-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 asΒ-catenin, c-myb, APC and SIAH1, for ubiquitin-mediate degradation. SIAH1 may function as a tumor suppressor gene. The study showed that SIAH1 expression 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 apoptosis through major alteration of mitosis. Other showed that SIAH1 was activated during apoptosis and tumor suppression in p53-dependent and-independent 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 various 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 JNK 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.
In our present study, we implored the expression of SIAH1 and Bim in breast tissues and analyzed the relationship between SIAH1 expression and clinical pathology parameters. In addition, we examined the overexpression or knockdown of SIAH1 had an effect on the expression of Bim, the activity of MAPK pathway, apoptosis and invasion in breast cancer cells.
Materials and Methods
1. Patients and specimens
A 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 invasive 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 underwent curative surgical resection without having chemotherapy or radiation therapy. Formalin-fixed paraffin-embedded 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-70℃immediately after resection until the extraction of protein and RNA.
2. Immunohistochemical study and result assessment
Four-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 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%=1,51-75%=2,≥76%=3) were assessed in at least 5 high power fields (×400 magnification). The scores of each sample were multiplied to give a final score of 0,1,2,3,4,6 or 9, and the tissues were finally determined as negative if score< 4; and positive expression if score≥4.
3. Cell culture
Human breast cancer cell lines MCF-7, MDA-MB-231, and human breast epithelial MCF-10A cell line were maintained in Dulbcco's Modifed Eagle Medium (DMEM) supplemented 10% fetal bovine serum in a humidified atmosphere with 5% of CO2.
4. Western blot
The protein was extracted with lysis buffer (150 mM NaCl,1%NP-40,0.1%SDS, 2μg/ml aprotinin,1 mM PMSF) for 1 hour at 4℃. The supernatants were centrifuged at 12000 rpm for 30 minutes at 4℃. The supernatants containing total protein were harvested. Aliquots containing 60μg of 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 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 p-actin (1:200) overnight at 4℃and followed by each corresponding second antibody at room temperature for 2 hour at 37℃. Then the results developed by ECL. 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 correspondingβ-actin band to determine the expression of the protein.
5. RT-PCR
Total RNA was isolated using Trizol Reagent according to the manufacturer's instructions. SIAH1 and Bim RT-PCRs were performed using a TaKaRa RNA PCR Kit (AMV) Ver.3.0 according to the manufacturer's protocol.β-actin served as an internal control. After electrophoresis, the PCR products were stained with ethidium bromide and analyzed using a Bioimaging system. Relative band intensities were determined using NIH image software.
6. Small RNA Interference
The small interfering RNA (siRNA) duplexes were synthesized and purified by Ji Kai. SIAH1 target sequence was as follows:1:5'-AACTCCTGCCTCCTTATGTA TTT-3'; 2:5'-GAUAGGAACACGCAAGCAA-3'; 3:5'-GUUGCAUGUAGUAACA CUA-3'. The nonsilencing siRNA 1 (control siRNA 1) sequence was as follows: 5'-AAGAGCCGTCAGACTGCTACA-3'. Bim target sequence was as follows: 1:5'-ACCGAGAAGGUAGACAAUU-3'; 2:5'-CUACCUCCCUACAGACAGA-3'; 3: 5'-CUACCUCCCUACAGACAGA-3'. The nonsilencing siRNA 2 (control siRNA 2) sequence was as follows:5'-CGUACGCGGAAUACUUCGA-3'. Considering the relative effectiveness and stability, SIAH1 siRNA 1 and Bim siRNA 1 were selected by comparing our pilot experiments.
7. Cell transfection
The 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/Bim 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) Assay
The transfected cells were seeded in 96-well plates (1×104 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 a microplate reader. A blank with dimethyl sulfoxide (DMSO) alone was measured and subtracted from all values. All the assays were done at least 3 times independently. The cell growth curve was analyzed.
9. Matrigel invasion assay
Cell invasive ability was examined using a 24-well Transwell with 8μm pore polycarbonate memberance inserts according to the manufacturer's protocol. The Matrigel (100μg/ml 1:7) which was precooled with serum-free DMEM was applied to the upper surface of the membranes. After transfection for 24 hours, cells were seeded on the upper chamber (5×104 cells/well) and incubated for 32 hours. Nontransfected cells served as the control. Cells that 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 and fixed with 75% ice-cold ethanol at 4℃overnight. Cells (1×107) were centrifuged at 1000 rpm 5 minutes, and were resuspended with 50μg/ml propidium iodide for 45 minutes in the dark before analysis. The percentages of cells in the different cell cycle phases were determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software.
11. Statistical analysis
All statistical calculations were performed by SPSS 13.0 for Windows software. P values less than 0.05 were considered statistically significant.
Results
1、SIAH1 was down-regulated in breast cancer tissues and cell, and inducing 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 results showed there was a decreasing tendency in positive rate of SIAH1 and Bim expression from normal breast tissues (NBT) (85.0% and 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 carcinoma (IDC) (28.4% and 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 and IDC. The Spearman's correlation test revealed that the SIAH1 expression was significantly positively associated with Bim expression in the 231 breast specimens (P< 0.001, rs=0.395). The clinicopathological data analysis showed that the expression of SIAH1 (P=0.025) and Bim (P=0.045) were significantly lower in poorly-differentiated tumors (gradeⅢ) than in well to moderately-differentiated tumors (gradeⅠandⅡ). The SIAH1 (P=0.018) and Bim (P=0.032) expression were significantly lower in advanced-stage tumors (stageⅢandⅣ) compared to early-stage tumors (stageⅠandⅡ).
(2) SIAH1 mRNA and protein expression were significantly lower in breast cancer tissues and cell lines
The 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 epithelial 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%, respectively). MDA-MB-231 and MCF-7 cells co-transfected pcDNA3-myc-SIAHl and Bim siRNA 1 (approximately 0.96%±0.18% and 0.01%±0.002%, respectively) showed lower apoptosis rate than cells transfected with pcDNA3-myc-SIAH1 (approximately 18.06%±3.35% and 19.27%±4.32%, respectively).
(4) SIAH1 inhibiting the invasive ability of breast cancer cells was independent of Bim:Invasion assays showed that both MDA-MB-231 and MCF-7 cells transfected with SIAH1 siRNA 1 had more cells (55.21±5.23 and 43.17±4.12, respectively, P< 0.05) invaded onto the lower surfaces of the Transwell filters than the cells transfected with control siRNA 1 (13.26±2.18 and 11.43±1.83, respectively) or untreated (15.45±2.36 and 11.69±1.62, respectively). In contrast, both MDA-MB-231 and MCF-7 cells transfected with pcDNA3-myc-SIAH1 had fewer cells (3.21±0.96 and 2.93±0.73, respectively, P<0.05) invaded onto the lower surfaces of the Transwell filters than the cells transfected with pcDNA3-myc (14.36±2.32 and 11.73±1.76, respectively) or untreated (15.45±2.36 and 11.69±1.62, respectively). MDA-MB-231 and MCF-7 cells co-transfected with pcDNA3-myc-SIAH1 and Bim siRNA 1 (3.06±0.89 and 2.09±0.68, respectively) had no significant difference with cells transfected with pcDNA3-myc-SIAH1 (3.21±0.96 and 2.93±0.73, respectively).
2、SIAH1 induced apoptosis by activation of JNK/Bim pathway and inhibited cell invasion by inactivation of ERK pathway in breast cancer cells
(1) SIAH1 regulated the expression of MAPKs in MDA-MB-231 and MCF-7 cell lines:Western blot results showed that overexpression of SIAH1 up-regulated the expression of P-JNK but down-regulated the expression of P-ERK. Meanwhile, SIAH1 siRNAs down-regulated the expression of P-JNK but up-regulated the expression of P-ERK, and SIAH1 siRNA 1 was the most effective in the three SIAH1 siRNAs. The expression of P-P38 had no change after the cells were transfected with pcDNA3-myc-SIAH1 or SIAH1 siRNAs
(2) Overexpression of SIAH1 induced apoptosis of MDA-MB-231 and MCF-7 cells by JNK/Bim pathway:Western blot results showed that overexpression of SIAH1 significantly up-regulated the expression of Bim and P-JNK, but SP600125 significantly inhibited overexpression of SIAH1-regulated the expression of Bim and P-JNK. The flow cytometry results showed that overexpression of SIAH1 significantly increased the apoptosis in MDA-MB-231 and MCF-7 cell lines (17.96% or 19.31%, respectively), but SP600125 significantly inhibited overexpression of SIAH1-induced apoptosis (0.05% or 0.02%, respectively).
(3) SIAH1 inhibited cell invasion through ERK pathway:Western blot results confirmed that the expression of P-ERK up-regulated by SIAH1 siRNA 1 was suppressed by ERK inhibitor PD98059. And invasion assays showed that both MDA-MB-231 and MCF-7 cells transfected with SIAH1 siRNA 1 (47.78±6.47 or 34.11±5.64, respectively, P<0.05) had greater number of cells which invaded onto the lower surfaces of the Transwell filters than cells transfected with SIAH1 siRNA 1 and meanwhile incubated with PD98059 (6.32±1.01 or 7.14±1.12, respectively, P<0.05).
(4) Both JNK and ERK pathway effected on SIAH1-associated cell growth inhibition:MTT assay results showed that the suppressed proliferation of the cancer cells transfected with pcDNA3-myc-SIAH1 was significantly reversed by SP600125. We also observed that the level of proliferation in cells transfected with SIAH1 siRNA 1 was significantly higher than those cells transfected with SIAH1 siRNA 1 and meanwhile incubation with PD98059.
Conclusion
1. SIAH1 functions as a tumor suppressor gene in the breast carcinogenesis and SIAH1 is significantly positively correlated with the expression of Bim. SIAH1 and Bim expression are significantly correlated with well to moderately-differentiated tumors and early-stage tumors.
2. SIAH1 induces apoptosis of MDA-MB-231 and MCF-7 cells by JNK/Bim pathway.
3. SIAH1 inhibiting the invasive ability of breast cancer cells is independent of Bim.
乳腺癌是女性一种常见的恶性肿瘤,已成为女性死亡的主要原因。乳腺癌的发生经历了以下几个阶段：正常—普通导管增生—不典型增生—导管原位癌—乳腺浸润性导管癌,探讨与乳腺癌动态发展过程相关的基因对治疗乳腺癌具有重要意义。
SIAH家族蛋白是果蝇属SINA蛋白的同系物,人类有两个高度保守的同源SINA:SIAH1和SIAH2, siahl定位于16q12号染色体,编码282个氨基酸,与果蝇属sina有76%的氨基酸相似,siah2定位于3q25号染色体,编码了324个氨基酸,与果蝇属sina有68%的同一性,与siahl有77%的同一性。然而SIAH1和SIAH2蛋白在N端有显著差别。SIAH1蛋白具有E3泛素连接酶的活性,可调节一些蛋白的泛素化和降解,这些蛋白包括B-catenin, c-myb, APC和SIAH1本身。SIAH1可作为一种肿瘤抑制因子发挥生物学作用,研究发现SIAH1在肝癌细胞系中低表达且过表达SIAH1后可诱导肝癌细胞的凋亡。乳腺癌细胞过表达SIAH1后可通过改变有丝分裂来抑制细胞生长和诱导细胞凋亡。还有研究表明SIAH1可以在p53依赖和独立的细胞凋亡和肿瘤抑制细胞模型中表达。
Bim (Bcl-2 interacting mediator of cell death)是Bcl-2家族中BH3-only亚家族的成员,是一种重要的凋亡调节蛋白。现已明确,Bim分子在一定的凋亡刺激下被激活,活化的Bim分子即可通过与Bcl-2/Bax的相互作用激活Bax,从而引起线粒体途径的细胞凋亡。
最近有研究发现SIAH1可能通过JNK/c-jun途径来调节细胞凋亡。JNK信号通路在细胞应激和细胞凋亡中起重要作用,且JNK可通过调控Bim的表达来诱导细胞凋亡。所以我们推测SIAH1可能通过JNK通路来调控Bim,从而诱导乳腺癌细胞的凋亡,但上述假设是否成立?其具体机制如何?这些问题需要我们进一步研究证实。
本研究目的旨在探讨乳腺组织中SIAH1和Bim的表达情况及与临床病理学参数的意义。在细胞水平上,通过过表达和干扰SIAH1,观察其对Bim表达,MAPK信号通路活性及对乳腺癌细胞凋亡和生长侵袭能力的影响。
材料与方法
1、乳腺组织
本研究选取了231例乳腺组织标本(包括40例正常乳腺组织,53例乳腺不典型增生组织,36例乳腺导管原位癌组织和102例乳腺浸润性导管癌组织),均来自中国医科大学附属第一临床学院2005-2008年肿瘤外科手术切除的标本,所有患者术前均未接受放、化疗。标本经4%中性甲醛固定,石蜡包埋,HE常规染色后明确诊断。其中部分癌旁乳腺组织(远离肿瘤至少5厘米)和癌组织离体后立即放入液氮后-70℃冰箱保存备用。
2、免疫组织化学染色及结果判定
标本用中性福尔马林溶液固定,石蜡包埋,制成4μm切片,经脱蜡,脱苯,水化后,采用链菌素抗生物素蛋白-过氧化物酶免疫组化法(S-P法)检测SIAH1和Bim蛋白表达。多克隆抗体SIAH1和Bim4℃孵育过夜。以PBS缓冲液代替抗为阴性对照。结果判定：SIAH1和Bim以细胞质中出现棕黄色颗粒为阳性显色。高倍视野(×400)下选阳性信号最强区域计数200个肿瘤细胞中阳性细胞数,按SIAH1和Bim表达百分率分为以下四个等级：0%为0分,1%-50%为1分,51%-75%为2分,>75%为3分。根据免疫组化染色强度分为三个等级：浅黄色计为1分,棕黄色计为2分,黄褐色计为3分。以阳性细胞率和染色强度的分值乘积作为每一例的积分,积分<4者判定为阴性,积分≥4为阳性。
3、细胞培养
用含10%新鲜胎牛血清的DMEM培养基,在37℃,5%CO2的条件下培养人乳腺癌细胞系MDA-MB-231和MCF7,人乳腺正常上皮细胞系MCF7-10A。
4、Western Blot
在收集的细胞内加入裂解液充分裂解,低温高速离心(4℃,12000转／min,30min),提取上清为总蛋白。上样蛋白量为60μg。电泳(12%SDS-PAGE凝胶)、转印、封闭,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β-actin (1:200),4℃孵育过夜,分别与各自对应的二抗(1：4000)室温孵育2小时,ECL显色,结果经自动凝胶成像分析仪采集,进行灰度值测定。
5、RT-PCR
采用TrizolTM试剂提取乳腺癌组织或培养细胞的总RNA,利用RNA PCR Kit (AMV)Ver.3.0试剂盒进行反转录。分别扩增SIAH1和Bim,以β-actin为内参。扩增产物经1.5%琼脂糖凝胶电泳后,成像分析。
6、siRNA干扰
根据siRNA设计原则,选取人SIAH1 mRNA和Bim mRNA中的特异性核苷酸片段为靶目标,应用Ambion公司在线siRNA设计软件设计SIAH1和Bim的RNAi序列,经Blast确定所选靶向的基因是特异的,序列由上海吉凯基因化学技术有限公司合成。实验分三组：空白对照组、非特异性siRNA转染组和特异性siRNA转染组,每个实验均重复三次,转染具体步骤按Lipofect 2000试剂说明书进行。SIAH1 siRNA序列：1:5'-AACTCCTGCCTCCTTATGTATTT-3',2:5'-GAUAGGAAC ACGCAAGCAA-3',3:5'-GUUGCAUGUAGUAACACUA-3'。非特异性siRNA 1 (control siRNA 1)序列：5'-AAGAGCCGTCAGACTGCTACA-3'。Bim siRNA序列：1:5'-ACCGAGAAGGUAGACAAUU-3',2:5'-CUACCUCCCUACAGACA GA-3',3:5'-CUACCUCCCUACAGACAGA-3'。非特异性siRNA 2 (control siRNA 2)序列：5'-CGUACGCGGAAUACUUCGA-3'。鉴于转染效率和稳定性,我们选择SIAH1 siRNA 1和Bim siRNA 1进行实验。
7、细胞转染
SIAH1表达质粒pcDNA3-myc-SIAH1和对照质粒pcDNA3-myc由Matsuzawa Shu-ichi (Burnham Institute, La Jolla, California, USA)教授惠赠。具体转染步骤按脂质体LipofectamineTM试剂说明书进行。以未转染的细胞和转染空载体细胞作为对照。
8、MTT法检测细胞增殖
将单个细胞悬液接种于96孔培养板中,每孔含104个细胞,培养24小时,每孔加入MTT (Methylthiazolyldiphenyl-tetrazolium bromide)溶液继续培养4小时,加入DMSO,490nm波长下测定各孔吸收值,以不含细胞的等体积培养基作对照。绘制细胞生长曲线。
9、细胞侵袭能力检测
在transwell小室下室加入600μl含10%胎牛血清DMEM培养基,上室加入100μl预冷的用无血清DMEM培养基稀释的Matrigel (1:7),将转染后24小时的细胞接种到上室。37℃,5%CO2孵箱中培养32小时后吸尽培养基,PBS清洗后,甲醇室温固定15分钟,用棉签擦掉微孔膜上表面的细胞,苏木素染色,室温干燥过夜。取下微孔膜,置载玻片上,镜下观察。
10、流式细胞仪检测细胞凋亡
收集对数生长期细胞制成1×107个／ml细胞悬液。取1ml细胞悬液75%冷乙醇于4℃固定、离心后制成500μl的细胞悬液,加碘化丙啶染液于4℃孵育45分钟后上机检测,ModFitLT3.0软件分析细胞凋亡。sub-G1期细胞代表凋亡细胞。
11、统计分析
各组资料利用SPSS for Window 13.0进行统计分析。P<0.05有统计学意义。
结果
1、SIAH1在乳腺癌中低表达,且可通过上调Bim表达来诱导乳腺癌细胞凋亡
(1) SIAH1和Bim蛋白在乳腺癌癌变过程中表达逐渐下调,并且二者表达与乳腺癌分期和分化相关：免疫组化结果显示,SIAH1和Bim蛋白在乳腺正常组织,乳腺不典型增生,乳腺导管原位癌和乳腺浸润性导管癌中的阳性表达率分别为85.0%与87.5%,66.0%与67.9%,52.8%与63.9%,28.4%and 34.3%。统计学结果显示：SIAH1 (P=0.002)和Bim (P=0.008)蛋白在乳腺正常组织和导管原位癌中表达有明显差别,SIAH1 (P=0.016)和Bim (P=0.002)蛋白在导管原位癌和浸润性导管癌中表达也有明显统计学意义。在231例乳腺组织中,SIAH1与Bim蛋白表达呈正相关(P<0.001,rs=0.395)。SIAH1 (P=0.025和P=0.018)与Bim(P=0.045和P=0.032)蛋白在高分化和早期乳腺癌中呈高表达。
(2) SIAH1 mRNA和蛋白在乳腺癌组织和乳腺癌细胞系中低表达：RT-PCR和Western blot结果显示,SIAH1 mRNA (P<0.05)与蛋白(P＜0.05)在乳腺癌组织表达低于癌旁乳腺组织,在乳腺癌细胞系MDA-MB-231和MCF-7中表达低于乳腺正常上皮细胞系MCF-10A (P<0.05)。
(3)过表达SIAH1后可上调Bim的表达,从而诱导乳腺癌细胞凋亡：实验结果表明,与乳腺癌细胞系MDA-MB-231和MCF-7转染空质粒组相比,转染pcDNA3-myc-SIAH1后,Bim(P＜0.05)表达上调,细胞凋亡率(P＜0.05)增加;与乳腺癌细胞系MDA-MB-231和MCF-7转染pcDNA3-myc-SIAH1相比,共转染pcDNA3-myc-SIAH1和SIAH1 siRNA 1后,凋亡率(P＜0.05)明显减少。
(4)过表达SIAH1以不依赖Bim的途径来抑制乳腺癌细胞的侵袭：细胞侵袭实验表明,与乳腺癌细胞系MDA-MB-231和MCF-7转染control siRNA 1组(13.26±2.18与11.43±1.83)或未转染组(15.45±2.36与11.69±1.62)相比,转染SIAH1 siRNA 1组(55.21±5.23与43.17±4.12)后,乳腺癌细胞侵袭力(P＜0.05)增加。相反的,乳腺癌细胞系MDA-MB-231和MCF-7转染pcDNA3-myc-SIAH1 (3.21±0.96与2.93±0.73)后,细胞侵袭力比转染pcDNA3-myc组(14.36±2.32与11.73±1.76)或未转染组(15.45±2.36与11.69±1.62)降低。乳腺癌细胞系MDA-MB-231和MCF-7共转染pcDNA3-myc-SIAH1与Bim siRNA 1 (3.06±0.89与2.09±0.68)后,细胞侵袭力与单独转染pcDNA3-myc-SIAH1组(3.21±0.96与2.93±0.73)相比无明显差别。
2、SIAH1可激活JNK通路上调Bim表达来诱导乳腺癌细胞凋亡,且可使ERK通路失活来抑制乳腺癌细胞侵袭
(1) SIAH1在乳腺癌细胞系中可调控MAPK通路的活性：Western blot结果显示,过表达SIAH1后可上调P-JNK蛋白的表达且可下调P-ERK蛋白的表达。相反的,干扰SIAH1后可下调P-JNK蛋白的表达且可上调P-ERK蛋白的表达。过表达或干扰SIAH1对P-P38蛋白的表达无明显影响。
(2)过表达SIAH1可通过激活JNK/Bim通路来诱导乳腺癌细胞凋亡：Western blot结果表明,采用JNK通路的特异性抑制剂SP600125后,过表达SIAH1导致的P-JNK和Bim蛋白表达上调被抑制。流式结果显示,过表达SIAH1诱导的乳腺癌细胞凋亡可被SP600125抑制。
(3)过表达SIAH1可通过ERK通路来抑制乳腺癌细胞侵袭：Western blot结果表明,采用ERK通路的特异性抑制剂PD98059后,干扰SIAH1导致的P-ERK蛋白表达上调被抑制。Transwell结果表明,干扰SIAH1导致的乳腺癌细胞侵袭力增加可被PD98059抑制。
(4)JNK通路和ERK通路同时影响SIAH1调节的乳腺癌细胞生长：MTT结果显示,过表达SIAH1抑制乳腺癌细胞的生长可被SP600125影响。同时,我们观察到干扰SIAH1促进乳腺癌细胞的生长可被PD98059抑制。
结论
1、SIAH1在乳腺癌癌变过程中可作为一种肿瘤抑制因子发挥作用,SIAH1与Bim表达呈正相关,并且它们均与乳腺癌的临床病理分期和分化密切相关。
2、在乳腺癌细胞中SIAH1通过调控JNK通路的活性来上调Bim表达,促进乳腺癌细胞凋亡。
3、在乳腺癌细胞中SIAH1通过调控ERK通路的活性来抑制乳腺癌细胞侵袭,且不依赖于Bim的表达。
Breast 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, 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) proteins are homologues of Drosophila seven in absentia (SINA) protein. SINA has two human homologues, SIAH1 and SIAH2. Siahl 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-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 asΒ-catenin, c-myb, APC and SIAH1, for ubiquitin-mediate degradation. SIAH1 may function as a tumor suppressor gene. The study showed that SIAH1 expression 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 apoptosis through major alteration of mitosis. Other showed that SIAH1 was activated during apoptosis and tumor suppression in p53-dependent and-independent 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 various 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 JNK 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.
In our present study, we implored the expression of SIAH1 and Bim in breast tissues and analyzed the relationship between SIAH1 expression and clinical pathology parameters. In addition, we examined the overexpression or knockdown of SIAH1 had an effect on the expression of Bim, the activity of MAPK pathway, apoptosis and invasion in breast cancer cells.
Materials and Methods
1. Patients and specimens
A 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 invasive 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 underwent curative surgical resection without having chemotherapy or radiation therapy. Formalin-fixed paraffin-embedded 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-70℃immediately after resection until the extraction of protein and RNA.
2. Immunohistochemical study and result assessment
Four-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 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%=1,51-75%=2,≥76%=3) were assessed in at least 5 high power fields (×400 magnification). The scores of each sample were multiplied to give a final score of 0,1,2,3,4,6 or 9, and the tissues were finally determined as negative if score< 4; and positive expression if score≥4.
3. Cell culture
Human breast cancer cell lines MCF-7, MDA-MB-231, and human breast epithelial MCF-10A cell line were maintained in Dulbcco's Modifed Eagle Medium (DMEM) supplemented 10% fetal bovine serum in a humidified atmosphere with 5% of CO2.
4. Western blot
The protein was extracted with lysis buffer (150 mM NaCl,1%NP-40,0.1%SDS, 2μg/ml aprotinin,1 mM PMSF) for 1 hour at 4℃. The supernatants were centrifuged at 12000 rpm for 30 minutes at 4℃. The supernatants containing total protein were harvested. Aliquots containing 60μg of 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 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 p-actin (1:200) overnight at 4℃and followed by each corresponding second antibody at room temperature for 2 hour at 37℃. Then the results developed by ECL. 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 correspondingβ-actin band to determine the expression of the protein.
5. RT-PCR
Total RNA was isolated using Trizol Reagent according to the manufacturer's instructions. SIAH1 and Bim RT-PCRs were performed using a TaKaRa RNA PCR Kit (AMV) Ver.3.0 according to the manufacturer's protocol.β-actin served as an internal control. After electrophoresis, the PCR products were stained with ethidium bromide and analyzed using a Bioimaging system. Relative band intensities were determined using NIH image software.
6. Small RNA Interference
The small interfering RNA (siRNA) duplexes were synthesized and purified by Ji Kai. SIAH1 target sequence was as follows:1:5'-AACTCCTGCCTCCTTATGTA TTT-3'; 2:5'-GAUAGGAACACGCAAGCAA-3'; 3:5'-GUUGCAUGUAGUAACA CUA-3'. The nonsilencing siRNA 1 (control siRNA 1) sequence was as follows: 5'-AAGAGCCGTCAGACTGCTACA-3'. Bim target sequence was as follows: 1:5'-ACCGAGAAGGUAGACAAUU-3'; 2:5'-CUACCUCCCUACAGACAGA-3'; 3: 5'-CUACCUCCCUACAGACAGA-3'. The nonsilencing siRNA 2 (control siRNA 2) sequence was as follows:5'-CGUACGCGGAAUACUUCGA-3'. Considering the relative effectiveness and stability, SIAH1 siRNA 1 and Bim siRNA 1 were selected by comparing our pilot experiments.
7. Cell transfection
The 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/Bim 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) Assay
The transfected cells were seeded in 96-well plates (1×104 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 a microplate reader. A blank with dimethyl sulfoxide (DMSO) alone was measured and subtracted from all values. All the assays were done at least 3 times independently. The cell growth curve was analyzed.
9. Matrigel invasion assay
Cell invasive ability was examined using a 24-well Transwell with 8μm pore polycarbonate memberance inserts according to the manufacturer's protocol. The Matrigel (100μg/ml 1:7) which was precooled with serum-free DMEM was applied to the upper surface of the membranes. After transfection for 24 hours, cells were seeded on the upper chamber (5×104 cells/well) and incubated for 32 hours. Nontransfected cells served as the control. Cells that 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 and fixed with 75% ice-cold ethanol at 4℃overnight. Cells (1×107) were centrifuged at 1000 rpm 5 minutes, and were resuspended with 50μg/ml propidium iodide for 45 minutes in the dark before analysis. The percentages of cells in the different cell cycle phases were determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software.
11. Statistical analysis
All statistical calculations were performed by SPSS 13.0 for Windows software. P values less than 0.05 were considered statistically significant.
Results
1、SIAH1 was down-regulated in breast cancer tissues and cell, and inducing 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 results showed there was a decreasing tendency in positive rate of SIAH1 and Bim expression from normal breast tissues (NBT) (85.0% and 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 carcinoma (IDC) (28.4% and 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 and IDC. The Spearman's correlation test revealed that the SIAH1 expression was significantly positively associated with Bim expression in the 231 breast specimens (P< 0.001, rs=0.395). The clinicopathological data analysis showed that the expression of SIAH1 (P=0.025) and Bim (P=0.045) were significantly lower in poorly-differentiated tumors (gradeⅢ) than in well to moderately-differentiated tumors (gradeⅠandⅡ). The SIAH1 (P=0.018) and Bim (P=0.032) expression were significantly lower in advanced-stage tumors (stageⅢandⅣ) compared to early-stage tumors (stageⅠandⅡ).
(2) SIAH1 mRNA and protein expression were significantly lower in breast cancer tissues and cell lines
The 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 epithelial 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%, respectively). MDA-MB-231 and MCF-7 cells co-transfected pcDNA3-myc-SIAHl and Bim siRNA 1 (approximately 0.96%±0.18% and 0.01%±0.002%, respectively) showed lower apoptosis rate than cells transfected with pcDNA3-myc-SIAH1 (approximately 18.06%±3.35% and 19.27%±4.32%, respectively).
(4) SIAH1 inhibiting the invasive ability of breast cancer cells was independent of Bim:Invasion assays showed that both MDA-MB-231 and MCF-7 cells transfected with SIAH1 siRNA 1 had more cells (55.21±5.23 and 43.17±4.12, respectively, P< 0.05) invaded onto the lower surfaces of the Transwell filters than the cells transfected with control siRNA 1 (13.26±2.18 and 11.43±1.83, respectively) or untreated (15.45±2.36 and 11.69±1.62, respectively). In contrast, both MDA-MB-231 and MCF-7 cells transfected with pcDNA3-myc-SIAH1 had fewer cells (3.21±0.96 and 2.93±0.73, respectively, P<0.05) invaded onto the lower surfaces of the Transwell filters than the cells transfected with pcDNA3-myc (14.36±2.32 and 11.73±1.76, respectively) or untreated (15.45±2.36 and 11.69±1.62, respectively). MDA-MB-231 and MCF-7 cells co-transfected with pcDNA3-myc-SIAH1 and Bim siRNA 1 (3.06±0.89 and 2.09±0.68, respectively) had no significant difference with cells transfected with pcDNA3-myc-SIAH1 (3.21±0.96 and 2.93±0.73, respectively).
2、SIAH1 induced apoptosis by activation of JNK/Bim pathway and inhibited cell invasion by inactivation of ERK pathway in breast cancer cells
(1) SIAH1 regulated the expression of MAPKs in MDA-MB-231 and MCF-7 cell lines:Western blot results showed that overexpression of SIAH1 up-regulated the expression of P-JNK but down-regulated the expression of P-ERK. Meanwhile, SIAH1 siRNAs down-regulated the expression of P-JNK but up-regulated the expression of P-ERK, and SIAH1 siRNA 1 was the most effective in the three SIAH1 siRNAs. The expression of P-P38 had no change after the cells were transfected with pcDNA3-myc-SIAH1 or SIAH1 siRNAs
(2) Overexpression of SIAH1 induced apoptosis of MDA-MB-231 and MCF-7 cells by JNK/Bim pathway:Western blot results showed that overexpression of SIAH1 significantly up-regulated the expression of Bim and P-JNK, but SP600125 significantly inhibited overexpression of SIAH1-regulated the expression of Bim and P-JNK. The flow cytometry results showed that overexpression of SIAH1 significantly increased the apoptosis in MDA-MB-231 and MCF-7 cell lines (17.96% or 19.31%, respectively), but SP600125 significantly inhibited overexpression of SIAH1-induced apoptosis (0.05% or 0.02%, respectively).
(3) SIAH1 inhibited cell invasion through ERK pathway:Western blot results confirmed that the expression of P-ERK up-regulated by SIAH1 siRNA 1 was suppressed by ERK inhibitor PD98059. And invasion assays showed that both MDA-MB-231 and MCF-7 cells transfected with SIAH1 siRNA 1 (47.78±6.47 or 34.11±5.64, respectively, P<0.05) had greater number of cells which invaded onto the lower surfaces of the Transwell filters than cells transfected with SIAH1 siRNA 1 and meanwhile incubated with PD98059 (6.32±1.01 or 7.14±1.12, respectively, P<0.05).
(4) Both JNK and ERK pathway effected on SIAH1-associated cell growth inhibition:MTT assay results showed that the suppressed proliferation of the cancer cells transfected with pcDNA3-myc-SIAH1 was significantly reversed by SP600125. We also observed that the level of proliferation in cells transfected with SIAH1 siRNA 1 was significantly higher than those cells transfected with SIAH1 siRNA 1 and meanwhile incubation with PD98059.
Conclusion
1. SIAH1 functions as a tumor suppressor gene in the breast carcinogenesis and SIAH1 is significantly positively correlated with the expression of Bim. SIAH1 and Bim expression are significantly correlated with well to moderately-differentiated tumors and early-stage tumors.
2. SIAH1 induces apoptosis of MDA-MB-231 and MCF-7 cells by JNK/Bim pathway.
3. SIAH1 inhibiting the invasive ability of breast cancer cells is independent of Bim.
引文
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