siRNA对乳腺癌特异性基因表达影响及与S100A4、ETS1、VEGF、CD105基因表达关系的研究
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摘要
乳腺癌是女性最常见的恶性肿瘤,全世界每年约有135万新增病例,42万人死于乳腺癌,我国乳腺癌的发病率正在逐年上升。乳腺癌浸润转移是患者预后差、死亡率高的关键原因,目前,乳腺癌治疗方法虽然有了极大的进展,但乳腺癌的生存率并没有明显提高,因此,从分子生物学上认识乳腺癌的发生发展的机制、导致浸涧转移的原因、寻求乳腺癌恶性转化的靶点及靶向治疗乳腺癌是当前研究的热点。
BCSG1 (breast cancer-specific gene 1)是一种特异的在乳腺癌中有选择性表达的基因,其定位于人类染色体10q23,DNA长度约5 kb。又称为Synucleinγ'(SNCG),与SNCs家族成员SNCG是同一基因,也可称spersyn。BCSG1基因编码合成1条含127个氨基酸的蛋白,它与雌性激素相关的乳腺和卵巢肿瘤密切相关。BCSG1表达有着特异时段性,其过度表达可能预示乳腺癌的恶性进程,是一种与乳腺癌进展有关的肿瘤标记物。研究发现,BCSG1的过度表达与乳腺癌的进程有关,是乳腺癌进展过程中的中晚期事件,BCSG1呈阳性表达的乳腺癌有更多浸润和转移的倾向。另有研究表明,BCSG1能够增强肿瘤细胞不利环境中的生存力,并能增强肿瘤的抗化疗药的能力,这也使得BCSG1成为治疗乳腺癌潜在的靶点。
RNA干扰(RNA interference, RNAi)是一种双链RNA(double-stranded RNA,dsRNA)分子在mRNA水平沉默相应序列基因的表达的过程,它通过21-23bp的dsRNA或发夹状RNA (hort hair pin RNAs, shRNAs)与特异性mRNA结合导致靶基因的降解,进而抑制目的基因的表达。RNAi技术以其快速、高效、操作简单等特点被广泛应用于基因功能的研究及疾病的基因治疗。
S100A4、Ets-1、VEGT及CD105标记的MVD均与肿瘤生长及浸润转移密切相关。
S-100A4是S-100钙离子结合蛋白超家族的成员之一,是一种有很高预后意义的肿瘤转移潜能分子标记物,主要分布于成纤维细胞、肌上皮细胞和肿瘤细胞的细胞浆和细胞外液中。可以促进细胞外基质的降解和重塑,增加肿瘤细胞的运动能力,抑制细胞凋亡,促进细胞异常增殖,促进血管生成,所以主要用于转移癌的研究。
研究证明部分癌基因是具有启动子特异性的转录调控因子,Ets家族是最大的信号依赖转录调控因子家族之一,Ets-1是近年来对肿瘤研究的一个热点的成员。Ets作为细胞转录调控因子家族在胚胎发育、组织成熟和指导时期特异性及组织特异性基因表达的过程中起着重要作用,其与肿瘤的发生也密切相关。Ets1即E26转录调节因子(transformation.specific 1, Ets-1, E26), Ets1基因位于染色体11q23-24的位置,编码分子量为54KD的蛋白质,其激活机制可能主要为扩增、染色体易位和重排。Ets1转录因子家族中的一员,其特点是同样拥有个独特的DNA结构域--Ets结构域。Ets1的DNA结合活性由激酶和转录因子控制,可通过调节细胞黏附、促进基质蛋白水解和血管生成以及提高肿瘤细胞缺氧耐受等机制参与多种肿瘤的侵袭和转移。
Ets-1与VEGF关系密切,有研究证明,Ets-1与VEGF及其受体Flt-1、Flk-1和neuropilin-1关系密切,Fit-K、Flk-1的启动子中包含Ets-1反应元件;血管生成素受体基因Tie-1、Tie-2和VEGF-R3也存在Ets结合位点。Watanabe等在培养的牛视网膜血管内皮细胞(BREC)研究证实缺氧导致VEGF上调Ets-1的表达,进而促进血管新生
VEGF是一类血管淋巴管内皮生长因子的总称,参与血管淋巴管生成的各个环节,是目前所知作用最强的一种促血管生长因子,参与血管和淋巴管形成的各个阶段,能特异结合血管内皮细胞,促进内皮细胞生长,并具有血管通透性,协助肿瘤细胞进入脉管系统,与肿瘤的转移和侵袭密切相关,也影响着肿瘤的疗效及预后。
肿瘤的生长和转移依赖于肿瘤血管生成,微血管密度(MVD)检测是评价肿瘤内血管生成的客观标准,MVD往往用CD105来表达的,CD105是一种由二硫键相连的同源二聚体的膜结合糖蛋白,分子量为180KD,由633个氨基酸组成,是TGF-β受体复合物的成分之一,CD105是肿瘤组织中新生血管内皮细胞的特异性标记物,其能区分新生血管和非新生血管,避免假阳性,从而获得正确评价肿瘤血管的生成能力。
RNA干扰(RNA interference, RNAi)是一种小分子双链RNA转录后的基因沉默。siRNA可特异地与序列互补的mRNA结合,促进其降解,从而在细胞内发挥基因抑制的作用,最终使细胞特定基因表达下调。1998年RNAi现象最先在线虫中发现,后来又在哺乳动物体内发现了RNAi现象,开创了RNAi技术应用于高等生物基因功能研究的先河。此后,RNAi技术逐渐成为生物医学研究领域中的一项重要的实验工具。RNAi因其可以高效、高特异的抑制靶基因的表达而迅速成为一种非常有效的在真核细胞中研究基因功能的工具,目前,RNAi技术已经被广泛应用于基因功能鉴定、基因表达转录后调控等热门研究领域,同时也为多种疾病特别是肿瘤的基因治疗提供了新的思路。
本研究中,为了研究BCSG1-siRNA对乳腺癌BCSG1表达及细胞侵袭力的影响,我们首先构建了BCSG1-siRNA质粒载体,转染乳腺癌MCF-7细胞,证明成功转染后,采用免疫细胞化学SP法及RT-PCR去检测各组细胞中BCSG1蛋白及mRNA的表达,采用平板克隆实验及Boyden chamber侵袭小室实验检测各组细胞的增殖及迁徙能力；另外,我们用转染BCSG1-siRNA的MCF-7细胞构建乳腺癌裸鼠种植瘤模型,采用免疫组织化学SP法及RT-PCR法检测裸鼠种植瘤组织中BCSG1蛋白及mRNA的表达；最后,我们采用免疫组化SP法检测了人乳腺癌及良性病变组织中BCSG1、S100A4、Ets-1、VEGF及CD105蛋白的表达。本研究从分子水平探讨乳腺癌的发生及浸润转移机制,为靶向治疗乳腺癌提供了理论基础和实验依据。
材料与方法
1.BCSG1-siRNA对乳腺癌MCF-7细胞BCSG-1基因表达及细胞侵袭性影响
人乳腺癌细胞MCF-7购自南京科诺生物有限公司。
乳腺癌MCF-7细胞的培养：乳腺癌MCF-7细胞以10%胎牛血清培养液(含青、链霉素各100u/ml)在37°C、5%CO2条件下贴壁生长培养。
BCSG1 siRNA序列的设计、合成及质粒载体的合成。
用脂质体将四条BCSG1-siRNA、一条无关siRNA转染MCF-7,G418抗性筛选、克隆扩增培养,建立稳定转染BCSG1-siRNA的细胞系。
实验分组：空脂质体组, (正常未转染)MCF-7对照组,阴性对照组,RCSG1-siRNA1转染组,BCSG1-siRNA2转染组,BCSG1-siRNA3转染组,BCSG1-siRNA4转染组。
采用免疫细胞化学SP法及RT-PCR法检测各组细胞中BCSG1蛋白及mRNA的表达。
根据免疫组织化学及RT-PCT结果选择对BCSG1抑制作用最强的BCSG1-siRNA转染的MCF-7细胞做平板克隆实验及Boyden chamber侵袭小室实验检测各组细胞的增殖及侵袭力。
2.BCSG1-siRNA对乳腺癌种植瘤组织中BCSG-1基因表达的影响
2.1两周龄BALB/c-nude (?)唯性裸鼠购自中国科学院上海斯莱克实验动物有限公司。无菌空气层流室内饲养,保持恒温(26-28℃)、恒湿(相对湿度40-60%)在SPF(specific pathogen Free)条件下进行饲养。
2.2实验分组：把裸鼠随即分为6组,每组5只。分别接种未转染的MCF7细胞组,接种转染无关序列的MCF7细胞组,接种转染BCSG1-iRNA-1的MCF7细胞组,接种转染BCSG1-siRNA-2的MCF7细胞组,接种转染BCSG1-siRNA-3的MCF7细胞组,接种转染BCSG1-siRNA-4的MCF7细胞组。
2.3构建食管癌裸鼠种植瘤模型
按实验分组要求,将各组MCF-7细胞接种到裸鼠皮下,接种2周左右,可见接种部位皮下陆续长出大小不等的硬结,为种植瘤模型建成。
2.4测量各组种植瘤瘤体体积及重量。
2.5采用免疫组织化学SP法及RT-PCR法检测各组种植瘤组织中BCSG1蛋白及mRNA的表达情况。
3.人乳腺组织中BCSG1的表达及与S100A4, ETS、VEGF和CD1 05的关系
3.1收集人乳腺纤维腺瘤40例,乳腺浸润性导管癌62例及其癌旁(正常及增生)组织48例,把浸润性导管癌组,按有无腋窝淋巴结转移分为1、2两组,其中1组13例,2组49例。
3.2采用免疫组织化学SP法检测各组BCSG1、S100A4, Ets1、VEGF及CD105蛋白的表达。
4.统计学处理
统计学处理均采用SPSS13.0统计软件,统计学数据用均数士标准差(X±S)表示,两组均数比较用t检验(t-test)；多组均数间的比较采用单因素方差分析(one-way ANOVA),多组均数间的两两比较采用LSD方法,检验水准α=0.05。
结果
1.细胞形态学观察
在倒置显微镜下观察,各对照组细胞均生长状态良好,细胞呈多边形贴壁生长,细胞间结构紧密,培养基中少许漂浮细胞；与对照组相比,各BCSG1-siRNA转染组,细胞体积变小,形态狭长,细胞间隙增大,细胞连接松散,部分细胞变圆、漂浮。
2.免疫细胞化学结果
BCGS-1蛋白免疫细胞化学阳性信号均呈棕黄色,位于细胞质。镜下每张爬片随机选取10个高倍视野,按阳性细胞数所占百分比及着色深浅进行结果判定。评分标准：(1)按阳性细胞数百分比计分：＜1%为0分；1%～25%为1分；26%～50%为2分；51%～75%为3分；＞76%为4分。(2)按阳性细胞着色深浅计分：无着色为0分；淡黄色为1分；棕黄色为2分；棕褐色为3分。最后综合两部分相乘为最后得分。BCSG-1-siRNA-1组为4.27±0.12,BCSG-1-siRNA-2组为4.19±0.22,BCSG-1-siRNA-3组为4.15±0.14,BCSG-1-siRNA-4组为4.17±0.13,无关序列组7.92±0.22,空白对照组8.02±0.13,MCF7对照组8.16±0.32。与各对照组相比,四组特异性siRNA转染组细胞中BCSG-1蛋白的表达均减弱,且差异均有统计学意义(ρ均＜0.05),但四组特异性siRNA转染组之间的差异无统计学意义(ρ＞0.05),各对照组之间无明显差异(ρ＞0.05)。
3.细胞RT-PCR结果
取5ul PCR扩增产物与2ul6×Loading Buffer混匀,点样于1%含溴化乙锭的琼脂糖凝胶内,80v电压下在1×TAE电泳缓冲液中电泳20～30min；凝胶成像系统拍照,记录扫描结果；使用Quantity One软件(BIO-RAD公司)测取各条带的灰度值,与内参β-actin比较计算各基因的相对表达水平。BCSG-1 mRNA在各组MCF-7细胞中均有表达,BCSG-1-siRNA-1组为0.624±0.010,BCSG-1-siRNA-2组为0.626±0.013,BCSG-1-siRNA-3组为0.634±0.008,BCSG-1-siRNA-4组为0.631±±0.010,无关序列组0.976±0.076,空白对照组0.983±0.052,MCF7对照组1.014±0.034。在四组特异性siRNA转染组织中的表达均明显低于各对照组(ρ＜0.05),且差异均有统计学意义(ρ＞0.05),四组特异性序列组之间的差异无统计学意义(ρ均＞0.05),各对照组对照组之间无明显差异(ρ＞0.05)。四组特异性siRNA与无关序列组及对照组相比,差异均有统计学意义(ρ＜0.05)。
4.平板克隆实验结果
选取转染对BCGS1抑制作用最强BCSG1-siRNA-3的细胞进行平板克隆实验,MCF-7细胞单细胞悬液分别接种于直径60mm的培养皿中,每皿分别含5×103,10×103及20×103个细胞。加入培养液,轻轻晃动,使细胞分散,细胞培养2-3周；当出现肉眼可见克隆时,终止培养；弃去培养液,PBS液洗3次；甲醇固定15min,使用苏木素染色2-3min,流水冲洗,空气干燥。镜下计数含50个细胞以上的克隆数。将3皿的克隆数折算克隆形成率,取均值。克隆形成率=克隆数/接种细胞数×100%。结果显示：MCF7对照组为32.33±2.52%,空脂质体组为28.33±3.51%,无关对照组为32.00±4.36%,BCSG1-siRNA-3组为4.17±0.13%。与各对照组相比,转染组克隆形成率明显高于各对照组,且差异均有统计学意义(ρ均＜0.05)。
5. Boyden chamber侵袭小室实验
用50ul无血清的RPMI-1640培养基稀释Matrigel胶,将其均匀铺盖于聚碳酸酯膜上,37℃放置30min使其成凝胶状,紫外灯照射30min备用；将细胞悬液稀释成浓度为5×105个/ml的单细胞悬液；每个小室加400ul稀释好的各组单细胞悬液,每组细胞加三个小室；常规条件下培养24h；取出小室,用棉签小心擦净膜上室面的胶和细胞,4%多聚甲醛固定,0.1%结晶紫染色后,细胞面朝上贴附到载玻片上；显微镜下随机计数5个高倍(400×)视野内的穿膜细胞数,计算平均值。实验结果表明：各组穿膜细胞数,MCF-7对照组为103.53±1.33,空脂质体组为100.93±1.81,无关对照组为101.20±1.71,BCSG1siRNA-3组为68.80±10.43。各干扰组与各对照组相比,穿膜细胞数有明显减少,且差异有统计学意义(ρ均＜0.05)。
6.各组裸鼠乳腺癌种植瘤生长情况比较
将六组细胞分别接种到各组裸鼠右前腋下皮下,两周后对照组裸鼠接种部位陆续长出大小不等的硬结,3周后各组裸鼠均陆续出现皮下结节,4周后,成瘤率达100%。裸鼠处死后,取出各组瘤体,分别称重,计算各组肿瘤生长抑制率(肿瘤抑制率=(对照组瘤重—实验组瘤重)/对照组瘤重×100%)。结果显示：两个对照组(A、B)的瘤体质量分别为122.34±6.79及116.04±5.20,四组特异性siRNA转染组(C、D、E、F)肿瘤组织的质量分别为20.12±0.85、21.44±1.43、20.28±0.94及20.96±1.42,后四组瘤体明显小于两对照组,应用统计学LSD方法进行比较；四组特异性siRNA转染组之间相互比较差异无统计学意义(P均＞0.05),前两各对照组之间也无明显差异(P＞0.05)。与各对照组相比,四组特异性siRNA转染组裸鼠种植瘤较小,且差异均有统计学意义(P均＜0.05)。各组肿瘤生长抑制率比较,四个特异性siRNA转染组的肿瘤抑制率明显小于无关对照组,且差异有统计学意义(P均＜0.05),但四组特异性siRNA转染组的肿瘤抑制率之间无明显差异(P均＞0.05)。
7.各组裸鼠乳腺癌种植瘤组织RT-PCR结果
种植瘤BCSG1每条mRNA阳性条带与内对照条带灰度值的比值以均数标准差(X±S)表示,BCSG1 mRNA在裸鼠种植瘤各组中均有表达,用内参β-actin的mRNA值测种植瘤组织中BCSG1 mRNA的表达与内参的灰度比。种植瘤组织中BCSG1 mRNA的表达与内参的灰度比：BCSG-1-siRNA-1组为1.28±0.36,BCSG-1-siRNA-2组为1.21±0.38,BCSG-1-siRNA-3组为1.21±0.47,BCSG-1-siRNA-4组为0.86±0.17,无关序列组2.19±0.37,MCF-7对照组2.62±0.68。无关序列组和MCF-7对照组的裸鼠种植瘤可见明显BCSG1 mRNA阳性条带,而在BCSG1-siRNA转染的4组中,仅有少量BCSG1较弱的mRNA阳性条带。其中在四组特异性siRNA转染组织中的表达明显低于无关序列组及MCF-7对照组,四组特异性序列组之间没有明显差异(P＞0.05),无关序列组和未转染MCF-7对照组之间无明显差异(P＞0.05)。无关序列组和未转染对照组与四组特异性siRNA转染组间比较有统计学意义(P＞0.05)。
8.各组种植瘤中BCSG1蛋白的表达
采用高清晰图像处理系统处理免疫组化图像,计算BCSG1蛋白的表达量：BCSG-1-siRNA-1组为8.06±0.21,BCSG-1-siRNA-2组为6.98±0.43,BCSG-1-siRNA-3组为6.98±0.72,BCSG-1-siRNA-4组为7.70±0.47,无关序列组16.56±0.86,MCF-7对照组17.90±1.38。应用统计学LSD方法进行比较,前四组之间相互比较无明显差异(ρ＞0.05),无关序列与未转染MCF-7对照组之间无明显差异(ρ＞0.05)。无关序列组和未转染MCF-7对照组与四组特异性siRNA转染组间比较有统计学意义(ρ＜0.05)。
9.人乳腺肿瘤中BCSG1蛋白的表达
在乳腺纤维腺瘤、浸导癌旁乳腺组织、浸导癌无腋窝淋巴结转移组1、浸导癌有腋窝淋巴结转移组2四组阳性表达率分别为2.50%、12.50%、69.23%、77.55%。人乳腺癌组织中BCSG1蛋白表达明显高于良性及正常(癌周)乳腺组织,且差异均有统计学意义(ρ＜0.05),乳腺良、恶性肿瘤四组阳性率比较差异有统计学意义(ρ＜0.05),和肿瘤病理分级呈正相关,随着癌浸润转移程度的增加阳性表达率也增高。
10.乳腺肿瘤中S100A4的表达
在乳腺纤维腺瘤、浸导癌旁乳腺组织、浸导癌无腋窝淋巴结转移组1、浸导癌有腋窝淋巴结转移组2四组阳性表达率分别为5.00%、6.25%、53.85%、59.18%,乳腺良、恶性肿瘤四组阳性率比较差异有统计学意义(ρ＜0.05),和肿瘤病理分级呈正相关,随着癌浸润转移程度的增加阳性表达率也增高。良性肿瘤之间或恶性肿瘤之间比较无显著性差异(ρ＞0.05)。
11.乳腺肿瘤中Ets1的表达
在乳腺纤维腺瘤、浸导癌旁乳腺组织、浸导癌无腋窝淋巴结转移组1、浸导癌有腋窝淋巴结转移组2四组阳性表达率分别为12.50%、25.00%、53.85%、61.22%,乳腺肿瘤四组阳性率比较差异有统计学意义(ρ＜0.05),浸导癌1组、2组之间比较无统计学意义(ρ＞0.05)。
12.乳腺肿瘤中VEGF的表达
在乳腺纤维腺瘤、浸导癌旁乳腺组织、浸导癌无腋窝淋巴结转移组1、浸导癌有腋窝淋巴结转移组2四组阳性表达率分别为10.00%、8.33%、53.85%、73.47%,阳性率比较差异有统计学意义(ρ＜0.05),和肿瘤病理分级呈正相关,随着癌浸润转移程度的增加阳性表达率也增高。良性肿瘤之间或恶性肿瘤之间比较无显著性差异(ρ＞0.05)。
13.乳腺肿瘤中CD105表达的MVD
在乳腺纤维腺瘤、导管原位癌、浸润性导管癌中,MVD值(X±S)分别为9.33±2.08,18.69±3.37,42.67±14.02。乳腺良、恶性肿瘤三组比较差异有统计学意义,两两比较差异仍有统计学意义(ρ＜0.05),浸润性导管癌MVD记数明显增多。
14.乳腺浸润性导管癌中BCSG-1与S100A4、VEGF、Ets-1表达的相关性
在乳腺62例浸润性导管癌中BCSG-1与S100A4、VEGF表达呈正相关,ρ＜0.05；浸润性导管癌中Ets-1与VEGF的表达也呈正相关,ρ＜0.001；浸润性导管癌中BCSG-1与Ets-1无明显相关,ρ＞0.05。
结论
1. BCSG1蛋白及mRNA在人乳腺癌细胞MCF-7细胞和乳腺癌种植瘤中均高表达。
2. BCSG1-siRNA可特异性抑制人乳腺癌细胞MCF-7细胞和乳腺癌种植瘤中BCSG-1基因蛋白表达及mRNA的水平。
3. BCSG1-siRNA可抑制人乳腺癌细胞MCF-7细胞的浸润性。提示BCSG1-siRNA治疗乳腺癌是可行的,为乳腺癌的治疗提供了新思路。
4. BCSG1-siRNA可抑制裸鼠抑制瘤的生长。
5. BCSG1在人乳腺癌组织中高表达,和肿瘤病理分级呈正相关,随着癌浸润转移程度的增加阳性表达率也增高。
6. S100A4、Ets1、VEGF、CD105在乳腺癌组织中均高表达,且和肿瘤病理分级呈正相关。在乳腺浸润性导管癌中BCSG-1与S100A4、VEGF表达呈正相关,Ets-1与VEGF的表达也呈正相关,BCSG-1与Ets-1无明显相关。
7.联合检测BCSG1、S100A4、Ets1、VEGF、C105的表达,与乳腺肿瘤发生、发展、预后密切相关,检测五指标明显增高,说明肿瘤的恶性潜能较高,易发生浸润、转移,估计预后较差。
Breast cancer is the most common malignancy in women. Worldwide, it is estimated that more than one million women are diagnosed with breast cancer per year, and more than 410,000 will die from the disease. In recent years, the incidence of breast cancer is increasing in China. And the occurrence of invasion and metastasis accounts for poor prognosis and high mortality. Although the treatment of breast cancer has been a great progress, the survival rate of breast cancer was not significantly increased increase. Therefore, it is current research focus to understand Breast Cancer pathogenesis in the molecular biology search of the targets of malignant transformation of breast cancer and target therapy of breast cancer.
BCSG1, breast cancer-specific gene 1, is a specific selective expression in breast cancer gene, localized to human chromosome 10q23, and the DNA length is about 5 kb. BCSG1 can also be called spersyn, the same gene family members SNCG with SNCs. BCSG1 gene can encode and syntheticen a protein sequence with 127 amino acids, which is closely related to breast and ovarian cancer associated with sex hormones. BCSG1 is a tumor marker related to progression of breast cancer and the expression of CBCSG1 has a especial period and it was over expression may indicate breast cancer malignant process.
S100A4, Ets-1, VEGT and MVD were associated with tumor angiogenesis, invasion and metastasis.
S-100A4, one of the S-100 calcium binding protein super families, is a potential molecular marker, which has a great significance on prognosis of tumor. S-100A4 mainly distributes in fibroblasts, myoepithelial cells, cytoplasm of tumor cells and extracellular fluid. It can promote extracellular matrix degradation and remodeling, increasing athletic ability of tumor cells, inhibit apoptosis, and promoting abnormal cell proliferation and angiogenesis. Therefore, it is mainly used in the research of metastatic cancer.
Ets1, E26 transcription factor, involves in a variety of tumor invasion and metastasis through the regulation of cell adhesion, hydrolysis and matrix proteins to promote angiogenesis and increase tumor cell hypoxia and other mechanisms. VEGF, a general kind of vascular endothelial growth factor, takes part in all aspects of lymphatic and vessel formation. As a vascular growth factor, it is the strongest. It can specifically bind vascular endothelial cells, promote endothelial cell growth, and help tumor cells enter the vasculature. It is closely related to tumor metastasis and invasion and also affects the efficacy and prognosis.
Tumor growth and metastasis depends on tumor angiogenesis. Tumor minute vessel density (MVD) assessed by CD 105 staining is the current gold standard for evaluating tumor angiogenesis in the clinic.It is a membrane-bound glycoprotein, a disulfide linked homodimer,180 KD, composed by 633 amino acids. CD 105 is one of TGF-βreceptor complex. CD105 is the specific marker of endothelial cells of no-newly-born vessels in tumor tissue, which can differentiate between newly- born vessels and no-newly-born vessels, to avoid false positives, resulting in correct evaluation tumor angiogenesis.
RNAi, RNA interference, is a kind of double-stranded RNA molecules, which can silence the expression of corresponding gene sequence of at the mRNA level. RNAi can result in the degradation of the target gene mRNA through the bind between 21-23nt of dsRNA or hairpin RNA with the specific mRNA, and then lead to the reduced expression of the purposed products. RNAi is widely used in the study of gene function and disease gene therapy because of its fast, reliable and easy-to-features.
To investigate the influence of BCSG-1 siRNA on gene expression of BCSG-1 in human breast cancer and cell invasion, BCSG1 siRNA vector was constructed and transfected MCF-7 breast cancer cells. The use of Immunohistochemistry(SP) and RT-PCR were used to detect the expressions of BCSG1 protein and mRNA in the cells; plate cloning experiments and Boyden chamber were used to detect cell proliferation and migration in each group cells. In addition, breast cancer xenograft model in nude mice were built with MCF-7 cells transfected BCSG1 siRNA immunohistochemistry and RT-PCR were used to detect the expressions of BCSG1 protein and mRNA in the tissue of nude mice transplanted tumor. Finally, immunohistochemical (SP way) was used to detect the expressions of BCSG1, S100A4, Ets-1, VEGT and CD 105 protein in the tissues of breast cancer and breast benign lesions. In this study, we have investigated the mechanism of invasion and metastasis of breast cancer mechanism in molecular level, which provides a theoretical basis and experimental evidence for targeted therapy of breast cancer.
Materials and methods
1. Effect of BCSG-1 siRNA on the expression of BCSG-1 gene and invasion
of breast cancer MCF-7 cells
Human breast cancer cells MCF-7 were purchased from Kono Bio Co. Ltd. of Nanjing.
MCF-7 cells were cultured in the RPMI-1640 medium containing 100g/L fetal bovine serum, at 37℃in a 5% CO2 humidified atmosphere.
BCSG1 siRNA sequence were designed, synthesized and the synthesis of plasmid vector.
Groups:The normal control group, Empty liposome, Negative control group, BCSG1 siRNA 1 group, BCSG1 siRNA2 group, BCSG1 siRNA3 group, BCSG1 siRNA4 group.
The immunocytochemistry and RT-PCR were used to detect the expression of BCSG1 protein and mRNA.
Plate cloning experiments and Boyden chamber were used to detect proliferation and invasion of MCF-7 cells transfected with the BCSG1 siRNA which inhibits MCF-7 cells according to the results of immunohistochemistry and RT-PCT.
2. Effect of BCSG-1 siRNA on the expression of BCSG-1 gene in tumor tissue of nude mice transplanted with human breast cancer cells
2.1 BALB/C nude mice, female,2 weeks old, were purchased from Hayes Lake Animal Company of limited liability. They were Reared in the conditions of SPF (specific pathogen Free), kept in constant humidity (relative humidity40-60%), constant temperature (26-28℃).
2.2 Groups:The mice were randomly divided into 6 groups,5 each group: group of the mice were inoculated with MCF-7 cells untransfected, group of the mice were inoculated with MCF-7 cells transfected nonsense siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG1 siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG2 siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG3 siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG4 siRNA sequences.
2.3 Construction of human breast cancer xenograft model in nude mice MCF-7 cells were inoculated subcutaneously into nude mice according to request.Sclerosis, grain of rice, was shown at injection site gradually after inoculating 2 weeks.
2.4 Tumor volume and weight each group were measured.
2.5 Immunohistochemical and RT-PCR were used to detect the expressions of BCSG1 protein and mRNA in nude mice transplanted tumor tissue.
3. The expressions of BCSG1,S100A4,ETS1,VEGF and CD105 protein in human breast cancer tissue.
3.1 Collecting 40 cases of breast fibroadenoma,62 cases of invasive ductal carcinoma, divided into 2 groups according to whether there are lymph node metastasis (respectively 13 and 49 cases), and 48 cases of adjacent, In which 1 group of 13 cases,49 cases of group 2.
3.2 Immunohistochemical was used to detect the expressions of BCSG1, S100A4, Ets1, VEGF and CD 105 protein.
4. Statistical analysis:
Statistical analysis:measurement data were presented as mean value±tandard error of the mean. The differences between two groups were determined with the Student's t test and LSD test. The differences among three or more groups were determined with a one-way ANOVA. Computations were performed using the SPSS 10.0 software. The cutoff for statistical significance was set as P＜0.05.
Results:
1 The changes of the cell morphology
The control group had normal cells morphology. Cells are irregular polygon, and have a clear outline. After BCSG1 siRNA transfection, compared with the control groups, some cells are shrunken, the size become smaller, the cell boundaries become blur, the gap become widened, a small amount of cells are suspended in the culture medium.
2 The immunocytochemistry results
Positive signal of BCGS-1 protein showed brown and located in the cytoplasm. Under the Microscope,10 high power fields were randomly selected, according to the percentage of positive cells and the color depth the results were determined. Rating criteria:(1) the scores according to the percentage of positive:"1%for 0; 1% to 25% for 1 point; 26% to 50%for 2 points; 51% to 75% for 3 points;> 76% to 4 points. (2) the scores according to the color depth of positive cells:no coloring is 0; yellow is 1; brown for 2; tan color is 3. Finally, multiply the final integrated score of two parts. The expressions of BCSG protein in cells of BCSG-1-siRNA-1 group was 4.27±0.12, that of BCSG-1-siRNA-2 group was 4.19±0.22, that of BCSG-1-siRNA-3 group was 4.15±0.14 and that of BCSG-1-siRNA-4 group was 4.17±0.13, independent sequence group was 7.92±0.22, control group was 8.02±0.13, MCF7 control group was 8.16±0.32. Compared with the control groups, the expression of BCSG-1 protein in four groups of specific siRNA transfected cells were decreased, and the differences were statistically significant (P all<0.05), but there was no significant difference four groups of specific siRNA transfection group each other (P>0.05), and there also no significant difference among the control groups, (P> 0.05).
3 Cell RT-PCR results
5μl PCR amplification products and 2μl 6 x Loading Buffer were mixed, spotting in 1%agarose containing ethidium bromide gel, electrophoresis for 20～30min (in 1 x TAE electrophoresis buffer, in 80v voltage; shooting with gel Imaging and recording the scan results; using Quantity One software (BIO-RAD company) to take measurements of the gray value of each band, compared with the calculation of internal referenceβ-actin and got the relative expression level of each gene. BCSG-1 mRNA was all expressed in the MCF-7 cells each group. The relative expression level of BCSG-1 mRNA in BCSG-1-siRNA-1 group was 0.624±0.010, that BCSG-1-siRNA-2 group was 0.626±0.013, BCSG-1-siRNA-3 group was 0.634±0.008, BCSG-1-siRNA-4 group was 0.631±0.010, independent of sequence group was 0.97±0.076, control group was 0.983±0.052, MCF7 control group was 1.014±0.034. Compared with the control groups, the expressions of BCSG-1 mRNA in the groups transfected the four specific siRNA were significantly lower (P all<0.05), and the differences were statistically significant (P all<0.05). There were no statistically significant between the groups transfected four group-specific sequence each other (P all> 0.05) and there no significant difference between the control groups (P> 0.05).
4 Plate cloning experiments results
Single cell suspension of MCF-7 cells were seeded in 60m2 Petri dish, each dish separately with 5×103,10×103 and 20×103 cells. Adding culture medium, gently shaking, the cells dispersed. After 2-3 weeks, terminating cultivation when there is visible cloning cluster. Washed 3 times with PBS solution; Fixed with methanol for 15min, and stained with hematoxylin for 2-3min, Rinsed with running water, dry in air. Colonies including at least 50 cells were counted under microscope. The number of clones 3 dish will be converted into cloning efficiency, and taking the mean. Cloning efficiency = clone number/inoculation cell number×100%. The results showed that:MCF7 control group was 32.33±.52%, empty liposome group was 28.33±3.51%, independent of the control group was 32.00±4.36%, BCSG1 siRNA-3 group was 4.17±0.13%. Compared with the control group, the formation of transfected clones was significantly higher than the control group, and differences were statistically significant (P all<0.05).
5 Boyden chamber experiments
Matrigel glue, diluted with 50μl serum free RPMI-1640 medium, was to uniform evenly blanketed in polycarbonate membrane, irradiated by Uv for 30min when it to become gelatinous after standing for 30min in air 37℃. The cell suspension was diluted to single-cell suspension which concentration was 5×105/ml. According to the requirement, each small room plus 400μl diluted single-cell suspension and each cell plus the three small rooms. They were cultured for 24h under normal conditions; then removing the small room, carefully wiping the film with a cotton swab, fixed with 4% paraformaldehyde,0.1% crystal violet staining, the cells attached to the slide on the side; 5 high power field of view (400×) were randomly selected under microscope, the counting their penetrating cells, Averaging the results. The results show that:penetrating cells in each group, MCF7 control group was 103.53±1.33, empty liposome group was 100.93±1.81, independent of the control group was 101.20±1.71, BCSG1siRNA-3 group was 68.80±10.43. The interference group and control group, the number of penetrating cells significantly decreased, and the difference was statistically significant (P<0.05).
6 The growth of nude mice implanted tumor in each group
The six groups of cells were inoculated into the subcutaneous of the right anterior axillary of nude mice in each group. Scleroma, the size of a grain of rice, gradually appeared at the inoculation site after 3 weeks in the control group, and that appeared in all the groups one week later. And then after 4 weeks, the tumor formation rate in each group is 100%. After the mice were killed, taking out the tumor in each group, weighing, and calculating the rate of tumor growth inhibition (tumor inhibition rate=(control group, tumor weight of a tumor weight of experimental group)/tumor weight of control group x 100%). The results showed:two control groups (A, B) of the tumor mass were 122.34±6.79 and 116.04±5.20, four specific siRNA transfection group (C, D, E, F) the quality of tumor tissue were 20.12±0.85, 21.44±1.43,20.28±0.94 and 20.96±1.42, after the tumor was less than four groups of two control groups, application of statistical methods to compare LSD; four specific siRNA transfection group was no statistical difference between Significance (P> 0.05), the first two control groups no significant difference between (P> 0.05). Compared with the control group, four groups of specific siRNA transfection group nude mice is small and the differences were statistically significant (P＜0.05). The rate of tumor growth inhibition compared to the four specific siRNA transfection group was significantly smaller than the tumor inhibition rate independent of the control group, and the difference was statistically significant (P＜0.05), but four specific siRNA transfection group Tumor inhibition was no significant difference between the rate (P> 0.05).
7 The expression of BCSG1 mRNA in each tumor tissue of nude mice transplanted with MCF-7 cells
The expressions of BCSG1 mRNA in tumor tissue of nude mice transplanted with MCF-7 cells were respectively:BCSG-1-siRNA-1 group was 1.28±0.36, BCSG-1-siRNA-2 group was 1.21±0.38, BCSG-1-siRNA-3 group 1.21±0.47, BCSG-1-siRNA-4 group was 0.86±.17, independent of sequence group 2.19±0.37, control group 2.62±0.68. The implanted tumor in nude mice in unrelated sequence group and blank control group showed obvious BCSG1 mRNA positive band, and that in each BCSG1-siRNA transfected group, only a small amount of mRNA BCSG1 weak positive bands. Compared with each controlled group, the expressions of BCSG1 mRNA were significantly lower (P<0.05). Compared by LSD method, there were no significant difference between groups transfected four specific sequence (P>0.05), the group transfected independent of sequence and the non-transfected control group (P> 0.05).
8 The expression of BCSG1 protein in each tumor tissue of nude mice transplanted wih MCF-7 cells
High-definition image processing system was used to analysis immunohisto chemical image. The results showed:BCSG-1-siRNA-1 group was 8.06±0.21, BCSG-1-siRNA-2 group was 6.98±0.43, BCSG-1-siRNA-3 group was 6.98±0.72, BCSG-1-siRNA-4 group was 7.70＝0.47, independent of sequence group 16.56±0.86, control group 17.90±1.38. Compared with each control group, the expressions of BCSG1 protein were significantly lower (P<0.05),and there were no significant difference between groups transfected four specific sequence (P> 0.05), the group transfected independent of sequence and the non-transfected control group (P> 0.05).
9 The expression of BCSG1 protein in human breast tumor tissue.
The positive rate of BCSG1 protein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma with axillary lymph node metastasis were 2.50%,12.50%, 69.23%, and 77.55%. BCSG1 protein in human breast cancer tissue and normal expression was significantly higher than that of benign (noncancerous) breast tissue, and the differences were statistically significant (P<0.05); the differences in positive rates between benign and malignant tumor were significant (P<0.05); the expression of BCSG1 protein was positively correlated with tumor grade, and increased with the increasing of the degree of tumor invasion and metastasis.
10 The expression of S100A4 protein in human breast tumor tissue.
The positive rate of S100A4 protein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma axillary lymph node metastasis were 5.00%,6.25%, 53.85%,59.18%. Compared with with the benign (noncancerous) breast tissue, the expression of S100A4 protein in human breast cancer tissue and normal expression were significantly higher, and the differences were statistically significant (P<0.05); the differences in positive rates between benign and malignant tumor were significant (p＜0.05).
11 The expression of Etsl protein in human breast tumor tissue.
The positive rate of Etslprotein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma with axillary lymph node metastasis were 12.50%,25.00% and 53.85%,61.22%. The difference in positive rate between the four groups was statistically significant (P<0.05). Compared with the invasive ductal carcinoma with axillary lymph node metastasis, the expression of S100A4 protein in ductal carcinoma without lymph node metastasis no significant changes (P>0.05)
12 The expression of VEGF protein in human breast tumor tissue.
The positive rate of VEGF protein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma axillary lymph node metastasis were 10.00%,8.33%, 53.85%,73.47%.Compared with the benign (noncancerous) breast tissue, the expression of VEGF protein in human breast cancer tissue and normal expression were significantly higher, and the differences were statistically significant (P <0.05);the differences in positive rates between benign and malignant tumor were significant (P<0.05).
13 The expression of CD105 protein in human breast tumor tissue.
MVD values (X±S) In breast fibroadenoma, ductal carcinoma in situ, invasive ductal carcinoma were 9.33±2.08,18.69±3.37,42.67±14.02. The differences between Benign and malignant was statistically significant (P<0.05), MVD in invasive ductal carcinoma increased significantly.
Conclusion
1. The expression of BCSG1 protein and mRNA in human breast cancer MCF-7 cells and in tumor transplants of human breast cancer cell line in nude mice were high.
2. BCSG1 siRNA can specifically inhibit the expressions of BCSG1 protein and mRNA in the human breast cancer MCF-7 cell line and in the tumor transplants of
human breast cancer cell line in nude mice.
3. BCSG1 siRNA can inhibit breast cancer MCF-7 cells invasive. It guess BCSG1 siRNA treating breast cancer is feasible and provides a new idea for the treatment of breast cancer.
4. BCSG1 siRNA can inhabit the growth of the tumor transplants of human breast cancer cell line in nude mice.
5. The expression of BCSG1 gene is high in human breast cancer.
6. The expression of BCSG1 gene was positively correlated to tumor pathological grade, and with the degree of cancer invasion and metastasis increase, the positive rate of BCSG1 increased.
7. The expression of S100A4、Ets1、VEGF and CD105 protein are all high. BCSG1, S100A4, Ets1, VEGF, CD 105 are closely related to the generate, development and prognosis of breast cancer, and there is great significance on breast cancer prognosis if these five factors are joint detected.
BCSG1 (breast cancer-specific gene 1)是一种特异的在乳腺癌中有选择性表达的基因,其定位于人类染色体10q23,DNA长度约5 kb。又称为Synucleinγ'(SNCG),与SNCs家族成员SNCG是同一基因,也可称spersyn。BCSG1基因编码合成1条含127个氨基酸的蛋白,它与雌性激素相关的乳腺和卵巢肿瘤密切相关。BCSG1表达有着特异时段性,其过度表达可能预示乳腺癌的恶性进程,是一种与乳腺癌进展有关的肿瘤标记物。研究发现,BCSG1的过度表达与乳腺癌的进程有关,是乳腺癌进展过程中的中晚期事件,BCSG1呈阳性表达的乳腺癌有更多浸润和转移的倾向。另有研究表明,BCSG1能够增强肿瘤细胞不利环境中的生存力,并能增强肿瘤的抗化疗药的能力,这也使得BCSG1成为治疗乳腺癌潜在的靶点。
RNA干扰(RNA interference, RNAi)是一种双链RNA(double-stranded RNA,dsRNA)分子在mRNA水平沉默相应序列基因的表达的过程,它通过21-23bp的dsRNA或发夹状RNA (hort hair pin RNAs, shRNAs)与特异性mRNA结合导致靶基因的降解,进而抑制目的基因的表达。RNAi技术以其快速、高效、操作简单等特点被广泛应用于基因功能的研究及疾病的基因治疗。
S100A4、Ets-1、VEGT及CD105标记的MVD均与肿瘤生长及浸润转移密切相关。
S-100A4是S-100钙离子结合蛋白超家族的成员之一,是一种有很高预后意义的肿瘤转移潜能分子标记物,主要分布于成纤维细胞、肌上皮细胞和肿瘤细胞的细胞浆和细胞外液中。可以促进细胞外基质的降解和重塑,增加肿瘤细胞的运动能力,抑制细胞凋亡,促进细胞异常增殖,促进血管生成,所以主要用于转移癌的研究。
研究证明部分癌基因是具有启动子特异性的转录调控因子,Ets家族是最大的信号依赖转录调控因子家族之一,Ets-1是近年来对肿瘤研究的一个热点的成员。Ets作为细胞转录调控因子家族在胚胎发育、组织成熟和指导时期特异性及组织特异性基因表达的过程中起着重要作用,其与肿瘤的发生也密切相关。Ets1即E26转录调节因子(transformation.specific 1, Ets-1, E26), Ets1基因位于染色体11q23-24的位置,编码分子量为54KD的蛋白质,其激活机制可能主要为扩增、染色体易位和重排。Ets1转录因子家族中的一员,其特点是同样拥有个独特的DNA结构域--Ets结构域。Ets1的DNA结合活性由激酶和转录因子控制,可通过调节细胞黏附、促进基质蛋白水解和血管生成以及提高肿瘤细胞缺氧耐受等机制参与多种肿瘤的侵袭和转移。
Ets-1与VEGF关系密切,有研究证明,Ets-1与VEGF及其受体Flt-1、Flk-1和neuropilin-1关系密切,Fit-K、Flk-1的启动子中包含Ets-1反应元件;血管生成素受体基因Tie-1、Tie-2和VEGF-R3也存在Ets结合位点。Watanabe等在培养的牛视网膜血管内皮细胞(BREC)研究证实缺氧导致VEGF上调Ets-1的表达,进而促进血管新生
VEGF是一类血管淋巴管内皮生长因子的总称,参与血管淋巴管生成的各个环节,是目前所知作用最强的一种促血管生长因子,参与血管和淋巴管形成的各个阶段,能特异结合血管内皮细胞,促进内皮细胞生长,并具有血管通透性,协助肿瘤细胞进入脉管系统,与肿瘤的转移和侵袭密切相关,也影响着肿瘤的疗效及预后。
肿瘤的生长和转移依赖于肿瘤血管生成,微血管密度(MVD)检测是评价肿瘤内血管生成的客观标准,MVD往往用CD105来表达的,CD105是一种由二硫键相连的同源二聚体的膜结合糖蛋白,分子量为180KD,由633个氨基酸组成,是TGF-β受体复合物的成分之一,CD105是肿瘤组织中新生血管内皮细胞的特异性标记物,其能区分新生血管和非新生血管,避免假阳性,从而获得正确评价肿瘤血管的生成能力。
RNA干扰(RNA interference, RNAi)是一种小分子双链RNA转录后的基因沉默。siRNA可特异地与序列互补的mRNA结合,促进其降解,从而在细胞内发挥基因抑制的作用,最终使细胞特定基因表达下调。1998年RNAi现象最先在线虫中发现,后来又在哺乳动物体内发现了RNAi现象,开创了RNAi技术应用于高等生物基因功能研究的先河。此后,RNAi技术逐渐成为生物医学研究领域中的一项重要的实验工具。RNAi因其可以高效、高特异的抑制靶基因的表达而迅速成为一种非常有效的在真核细胞中研究基因功能的工具,目前,RNAi技术已经被广泛应用于基因功能鉴定、基因表达转录后调控等热门研究领域,同时也为多种疾病特别是肿瘤的基因治疗提供了新的思路。
本研究中,为了研究BCSG1-siRNA对乳腺癌BCSG1表达及细胞侵袭力的影响,我们首先构建了BCSG1-siRNA质粒载体,转染乳腺癌MCF-7细胞,证明成功转染后,采用免疫细胞化学SP法及RT-PCR去检测各组细胞中BCSG1蛋白及mRNA的表达,采用平板克隆实验及Boyden chamber侵袭小室实验检测各组细胞的增殖及迁徙能力；另外,我们用转染BCSG1-siRNA的MCF-7细胞构建乳腺癌裸鼠种植瘤模型,采用免疫组织化学SP法及RT-PCR法检测裸鼠种植瘤组织中BCSG1蛋白及mRNA的表达；最后,我们采用免疫组化SP法检测了人乳腺癌及良性病变组织中BCSG1、S100A4、Ets-1、VEGF及CD105蛋白的表达。本研究从分子水平探讨乳腺癌的发生及浸润转移机制,为靶向治疗乳腺癌提供了理论基础和实验依据。
材料与方法
1.BCSG1-siRNA对乳腺癌MCF-7细胞BCSG-1基因表达及细胞侵袭性影响
人乳腺癌细胞MCF-7购自南京科诺生物有限公司。
乳腺癌MCF-7细胞的培养：乳腺癌MCF-7细胞以10%胎牛血清培养液(含青、链霉素各100u/ml)在37°C、5%CO2条件下贴壁生长培养。
BCSG1 siRNA序列的设计、合成及质粒载体的合成。
用脂质体将四条BCSG1-siRNA、一条无关siRNA转染MCF-7,G418抗性筛选、克隆扩增培养,建立稳定转染BCSG1-siRNA的细胞系。
实验分组：空脂质体组, (正常未转染)MCF-7对照组,阴性对照组,RCSG1-siRNA1转染组,BCSG1-siRNA2转染组,BCSG1-siRNA3转染组,BCSG1-siRNA4转染组。
采用免疫细胞化学SP法及RT-PCR法检测各组细胞中BCSG1蛋白及mRNA的表达。
根据免疫组织化学及RT-PCT结果选择对BCSG1抑制作用最强的BCSG1-siRNA转染的MCF-7细胞做平板克隆实验及Boyden chamber侵袭小室实验检测各组细胞的增殖及侵袭力。
2.BCSG1-siRNA对乳腺癌种植瘤组织中BCSG-1基因表达的影响
2.1两周龄BALB/c-nude (?)唯性裸鼠购自中国科学院上海斯莱克实验动物有限公司。无菌空气层流室内饲养,保持恒温(26-28℃)、恒湿(相对湿度40-60%)在SPF(specific pathogen Free)条件下进行饲养。
2.2实验分组：把裸鼠随即分为6组,每组5只。分别接种未转染的MCF7细胞组,接种转染无关序列的MCF7细胞组,接种转染BCSG1-iRNA-1的MCF7细胞组,接种转染BCSG1-siRNA-2的MCF7细胞组,接种转染BCSG1-siRNA-3的MCF7细胞组,接种转染BCSG1-siRNA-4的MCF7细胞组。
2.3构建食管癌裸鼠种植瘤模型
按实验分组要求,将各组MCF-7细胞接种到裸鼠皮下,接种2周左右,可见接种部位皮下陆续长出大小不等的硬结,为种植瘤模型建成。
2.4测量各组种植瘤瘤体体积及重量。
2.5采用免疫组织化学SP法及RT-PCR法检测各组种植瘤组织中BCSG1蛋白及mRNA的表达情况。
3.人乳腺组织中BCSG1的表达及与S100A4, ETS、VEGF和CD1 05的关系
3.1收集人乳腺纤维腺瘤40例,乳腺浸润性导管癌62例及其癌旁(正常及增生)组织48例,把浸润性导管癌组,按有无腋窝淋巴结转移分为1、2两组,其中1组13例,2组49例。
3.2采用免疫组织化学SP法检测各组BCSG1、S100A4, Ets1、VEGF及CD105蛋白的表达。
4.统计学处理
统计学处理均采用SPSS13.0统计软件,统计学数据用均数士标准差(X±S)表示,两组均数比较用t检验(t-test)；多组均数间的比较采用单因素方差分析(one-way ANOVA),多组均数间的两两比较采用LSD方法,检验水准α=0.05。
结果
1.细胞形态学观察
在倒置显微镜下观察,各对照组细胞均生长状态良好,细胞呈多边形贴壁生长,细胞间结构紧密,培养基中少许漂浮细胞；与对照组相比,各BCSG1-siRNA转染组,细胞体积变小,形态狭长,细胞间隙增大,细胞连接松散,部分细胞变圆、漂浮。
2.免疫细胞化学结果
BCGS-1蛋白免疫细胞化学阳性信号均呈棕黄色,位于细胞质。镜下每张爬片随机选取10个高倍视野,按阳性细胞数所占百分比及着色深浅进行结果判定。评分标准：(1)按阳性细胞数百分比计分：＜1%为0分；1%～25%为1分；26%～50%为2分；51%～75%为3分；＞76%为4分。(2)按阳性细胞着色深浅计分：无着色为0分；淡黄色为1分；棕黄色为2分；棕褐色为3分。最后综合两部分相乘为最后得分。BCSG-1-siRNA-1组为4.27±0.12,BCSG-1-siRNA-2组为4.19±0.22,BCSG-1-siRNA-3组为4.15±0.14,BCSG-1-siRNA-4组为4.17±0.13,无关序列组7.92±0.22,空白对照组8.02±0.13,MCF7对照组8.16±0.32。与各对照组相比,四组特异性siRNA转染组细胞中BCSG-1蛋白的表达均减弱,且差异均有统计学意义(ρ均＜0.05),但四组特异性siRNA转染组之间的差异无统计学意义(ρ＞0.05),各对照组之间无明显差异(ρ＞0.05)。
3.细胞RT-PCR结果
取5ul PCR扩增产物与2ul6×Loading Buffer混匀,点样于1%含溴化乙锭的琼脂糖凝胶内,80v电压下在1×TAE电泳缓冲液中电泳20～30min；凝胶成像系统拍照,记录扫描结果；使用Quantity One软件(BIO-RAD公司)测取各条带的灰度值,与内参β-actin比较计算各基因的相对表达水平。BCSG-1 mRNA在各组MCF-7细胞中均有表达,BCSG-1-siRNA-1组为0.624±0.010,BCSG-1-siRNA-2组为0.626±0.013,BCSG-1-siRNA-3组为0.634±0.008,BCSG-1-siRNA-4组为0.631±±0.010,无关序列组0.976±0.076,空白对照组0.983±0.052,MCF7对照组1.014±0.034。在四组特异性siRNA转染组织中的表达均明显低于各对照组(ρ＜0.05),且差异均有统计学意义(ρ＞0.05),四组特异性序列组之间的差异无统计学意义(ρ均＞0.05),各对照组对照组之间无明显差异(ρ＞0.05)。四组特异性siRNA与无关序列组及对照组相比,差异均有统计学意义(ρ＜0.05)。
4.平板克隆实验结果
选取转染对BCGS1抑制作用最强BCSG1-siRNA-3的细胞进行平板克隆实验,MCF-7细胞单细胞悬液分别接种于直径60mm的培养皿中,每皿分别含5×103,10×103及20×103个细胞。加入培养液,轻轻晃动,使细胞分散,细胞培养2-3周；当出现肉眼可见克隆时,终止培养；弃去培养液,PBS液洗3次；甲醇固定15min,使用苏木素染色2-3min,流水冲洗,空气干燥。镜下计数含50个细胞以上的克隆数。将3皿的克隆数折算克隆形成率,取均值。克隆形成率=克隆数/接种细胞数×100%。结果显示：MCF7对照组为32.33±2.52%,空脂质体组为28.33±3.51%,无关对照组为32.00±4.36%,BCSG1-siRNA-3组为4.17±0.13%。与各对照组相比,转染组克隆形成率明显高于各对照组,且差异均有统计学意义(ρ均＜0.05)。
5. Boyden chamber侵袭小室实验
用50ul无血清的RPMI-1640培养基稀释Matrigel胶,将其均匀铺盖于聚碳酸酯膜上,37℃放置30min使其成凝胶状,紫外灯照射30min备用；将细胞悬液稀释成浓度为5×105个/ml的单细胞悬液；每个小室加400ul稀释好的各组单细胞悬液,每组细胞加三个小室；常规条件下培养24h；取出小室,用棉签小心擦净膜上室面的胶和细胞,4%多聚甲醛固定,0.1%结晶紫染色后,细胞面朝上贴附到载玻片上；显微镜下随机计数5个高倍(400×)视野内的穿膜细胞数,计算平均值。实验结果表明：各组穿膜细胞数,MCF-7对照组为103.53±1.33,空脂质体组为100.93±1.81,无关对照组为101.20±1.71,BCSG1siRNA-3组为68.80±10.43。各干扰组与各对照组相比,穿膜细胞数有明显减少,且差异有统计学意义(ρ均＜0.05)。
6.各组裸鼠乳腺癌种植瘤生长情况比较
将六组细胞分别接种到各组裸鼠右前腋下皮下,两周后对照组裸鼠接种部位陆续长出大小不等的硬结,3周后各组裸鼠均陆续出现皮下结节,4周后,成瘤率达100%。裸鼠处死后,取出各组瘤体,分别称重,计算各组肿瘤生长抑制率(肿瘤抑制率=(对照组瘤重—实验组瘤重)/对照组瘤重×100%)。结果显示：两个对照组(A、B)的瘤体质量分别为122.34±6.79及116.04±5.20,四组特异性siRNA转染组(C、D、E、F)肿瘤组织的质量分别为20.12±0.85、21.44±1.43、20.28±0.94及20.96±1.42,后四组瘤体明显小于两对照组,应用统计学LSD方法进行比较；四组特异性siRNA转染组之间相互比较差异无统计学意义(P均＞0.05),前两各对照组之间也无明显差异(P＞0.05)。与各对照组相比,四组特异性siRNA转染组裸鼠种植瘤较小,且差异均有统计学意义(P均＜0.05)。各组肿瘤生长抑制率比较,四个特异性siRNA转染组的肿瘤抑制率明显小于无关对照组,且差异有统计学意义(P均＜0.05),但四组特异性siRNA转染组的肿瘤抑制率之间无明显差异(P均＞0.05)。
7.各组裸鼠乳腺癌种植瘤组织RT-PCR结果
种植瘤BCSG1每条mRNA阳性条带与内对照条带灰度值的比值以均数标准差(X±S)表示,BCSG1 mRNA在裸鼠种植瘤各组中均有表达,用内参β-actin的mRNA值测种植瘤组织中BCSG1 mRNA的表达与内参的灰度比。种植瘤组织中BCSG1 mRNA的表达与内参的灰度比：BCSG-1-siRNA-1组为1.28±0.36,BCSG-1-siRNA-2组为1.21±0.38,BCSG-1-siRNA-3组为1.21±0.47,BCSG-1-siRNA-4组为0.86±0.17,无关序列组2.19±0.37,MCF-7对照组2.62±0.68。无关序列组和MCF-7对照组的裸鼠种植瘤可见明显BCSG1 mRNA阳性条带,而在BCSG1-siRNA转染的4组中,仅有少量BCSG1较弱的mRNA阳性条带。其中在四组特异性siRNA转染组织中的表达明显低于无关序列组及MCF-7对照组,四组特异性序列组之间没有明显差异(P＞0.05),无关序列组和未转染MCF-7对照组之间无明显差异(P＞0.05)。无关序列组和未转染对照组与四组特异性siRNA转染组间比较有统计学意义(P＞0.05)。
8.各组种植瘤中BCSG1蛋白的表达
采用高清晰图像处理系统处理免疫组化图像,计算BCSG1蛋白的表达量：BCSG-1-siRNA-1组为8.06±0.21,BCSG-1-siRNA-2组为6.98±0.43,BCSG-1-siRNA-3组为6.98±0.72,BCSG-1-siRNA-4组为7.70±0.47,无关序列组16.56±0.86,MCF-7对照组17.90±1.38。应用统计学LSD方法进行比较,前四组之间相互比较无明显差异(ρ＞0.05),无关序列与未转染MCF-7对照组之间无明显差异(ρ＞0.05)。无关序列组和未转染MCF-7对照组与四组特异性siRNA转染组间比较有统计学意义(ρ＜0.05)。
9.人乳腺肿瘤中BCSG1蛋白的表达
在乳腺纤维腺瘤、浸导癌旁乳腺组织、浸导癌无腋窝淋巴结转移组1、浸导癌有腋窝淋巴结转移组2四组阳性表达率分别为2.50%、12.50%、69.23%、77.55%。人乳腺癌组织中BCSG1蛋白表达明显高于良性及正常(癌周)乳腺组织,且差异均有统计学意义(ρ＜0.05),乳腺良、恶性肿瘤四组阳性率比较差异有统计学意义(ρ＜0.05),和肿瘤病理分级呈正相关,随着癌浸润转移程度的增加阳性表达率也增高。
10.乳腺肿瘤中S100A4的表达
在乳腺纤维腺瘤、浸导癌旁乳腺组织、浸导癌无腋窝淋巴结转移组1、浸导癌有腋窝淋巴结转移组2四组阳性表达率分别为5.00%、6.25%、53.85%、59.18%,乳腺良、恶性肿瘤四组阳性率比较差异有统计学意义(ρ＜0.05),和肿瘤病理分级呈正相关,随着癌浸润转移程度的增加阳性表达率也增高。良性肿瘤之间或恶性肿瘤之间比较无显著性差异(ρ＞0.05)。
11.乳腺肿瘤中Ets1的表达
在乳腺纤维腺瘤、浸导癌旁乳腺组织、浸导癌无腋窝淋巴结转移组1、浸导癌有腋窝淋巴结转移组2四组阳性表达率分别为12.50%、25.00%、53.85%、61.22%,乳腺肿瘤四组阳性率比较差异有统计学意义(ρ＜0.05),浸导癌1组、2组之间比较无统计学意义(ρ＞0.05)。
12.乳腺肿瘤中VEGF的表达
在乳腺纤维腺瘤、浸导癌旁乳腺组织、浸导癌无腋窝淋巴结转移组1、浸导癌有腋窝淋巴结转移组2四组阳性表达率分别为10.00%、8.33%、53.85%、73.47%,阳性率比较差异有统计学意义(ρ＜0.05),和肿瘤病理分级呈正相关,随着癌浸润转移程度的增加阳性表达率也增高。良性肿瘤之间或恶性肿瘤之间比较无显著性差异(ρ＞0.05)。
13.乳腺肿瘤中CD105表达的MVD
在乳腺纤维腺瘤、导管原位癌、浸润性导管癌中,MVD值(X±S)分别为9.33±2.08,18.69±3.37,42.67±14.02。乳腺良、恶性肿瘤三组比较差异有统计学意义,两两比较差异仍有统计学意义(ρ＜0.05),浸润性导管癌MVD记数明显增多。
14.乳腺浸润性导管癌中BCSG-1与S100A4、VEGF、Ets-1表达的相关性
在乳腺62例浸润性导管癌中BCSG-1与S100A4、VEGF表达呈正相关,ρ＜0.05；浸润性导管癌中Ets-1与VEGF的表达也呈正相关,ρ＜0.001；浸润性导管癌中BCSG-1与Ets-1无明显相关,ρ＞0.05。
结论
1. BCSG1蛋白及mRNA在人乳腺癌细胞MCF-7细胞和乳腺癌种植瘤中均高表达。
2. BCSG1-siRNA可特异性抑制人乳腺癌细胞MCF-7细胞和乳腺癌种植瘤中BCSG-1基因蛋白表达及mRNA的水平。
3. BCSG1-siRNA可抑制人乳腺癌细胞MCF-7细胞的浸润性。提示BCSG1-siRNA治疗乳腺癌是可行的,为乳腺癌的治疗提供了新思路。
4. BCSG1-siRNA可抑制裸鼠抑制瘤的生长。
5. BCSG1在人乳腺癌组织中高表达,和肿瘤病理分级呈正相关,随着癌浸润转移程度的增加阳性表达率也增高。
6. S100A4、Ets1、VEGF、CD105在乳腺癌组织中均高表达,且和肿瘤病理分级呈正相关。在乳腺浸润性导管癌中BCSG-1与S100A4、VEGF表达呈正相关,Ets-1与VEGF的表达也呈正相关,BCSG-1与Ets-1无明显相关。
7.联合检测BCSG1、S100A4、Ets1、VEGF、C105的表达,与乳腺肿瘤发生、发展、预后密切相关,检测五指标明显增高,说明肿瘤的恶性潜能较高,易发生浸润、转移,估计预后较差。
Breast cancer is the most common malignancy in women. Worldwide, it is estimated that more than one million women are diagnosed with breast cancer per year, and more than 410,000 will die from the disease. In recent years, the incidence of breast cancer is increasing in China. And the occurrence of invasion and metastasis accounts for poor prognosis and high mortality. Although the treatment of breast cancer has been a great progress, the survival rate of breast cancer was not significantly increased increase. Therefore, it is current research focus to understand Breast Cancer pathogenesis in the molecular biology search of the targets of malignant transformation of breast cancer and target therapy of breast cancer.
BCSG1, breast cancer-specific gene 1, is a specific selective expression in breast cancer gene, localized to human chromosome 10q23, and the DNA length is about 5 kb. BCSG1 can also be called spersyn, the same gene family members SNCG with SNCs. BCSG1 gene can encode and syntheticen a protein sequence with 127 amino acids, which is closely related to breast and ovarian cancer associated with sex hormones. BCSG1 is a tumor marker related to progression of breast cancer and the expression of CBCSG1 has a especial period and it was over expression may indicate breast cancer malignant process.
S100A4, Ets-1, VEGT and MVD were associated with tumor angiogenesis, invasion and metastasis.
S-100A4, one of the S-100 calcium binding protein super families, is a potential molecular marker, which has a great significance on prognosis of tumor. S-100A4 mainly distributes in fibroblasts, myoepithelial cells, cytoplasm of tumor cells and extracellular fluid. It can promote extracellular matrix degradation and remodeling, increasing athletic ability of tumor cells, inhibit apoptosis, and promoting abnormal cell proliferation and angiogenesis. Therefore, it is mainly used in the research of metastatic cancer.
Ets1, E26 transcription factor, involves in a variety of tumor invasion and metastasis through the regulation of cell adhesion, hydrolysis and matrix proteins to promote angiogenesis and increase tumor cell hypoxia and other mechanisms. VEGF, a general kind of vascular endothelial growth factor, takes part in all aspects of lymphatic and vessel formation. As a vascular growth factor, it is the strongest. It can specifically bind vascular endothelial cells, promote endothelial cell growth, and help tumor cells enter the vasculature. It is closely related to tumor metastasis and invasion and also affects the efficacy and prognosis.
Tumor growth and metastasis depends on tumor angiogenesis. Tumor minute vessel density (MVD) assessed by CD 105 staining is the current gold standard for evaluating tumor angiogenesis in the clinic.It is a membrane-bound glycoprotein, a disulfide linked homodimer,180 KD, composed by 633 amino acids. CD 105 is one of TGF-βreceptor complex. CD105 is the specific marker of endothelial cells of no-newly-born vessels in tumor tissue, which can differentiate between newly- born vessels and no-newly-born vessels, to avoid false positives, resulting in correct evaluation tumor angiogenesis.
RNAi, RNA interference, is a kind of double-stranded RNA molecules, which can silence the expression of corresponding gene sequence of at the mRNA level. RNAi can result in the degradation of the target gene mRNA through the bind between 21-23nt of dsRNA or hairpin RNA with the specific mRNA, and then lead to the reduced expression of the purposed products. RNAi is widely used in the study of gene function and disease gene therapy because of its fast, reliable and easy-to-features.
To investigate the influence of BCSG-1 siRNA on gene expression of BCSG-1 in human breast cancer and cell invasion, BCSG1 siRNA vector was constructed and transfected MCF-7 breast cancer cells. The use of Immunohistochemistry(SP) and RT-PCR were used to detect the expressions of BCSG1 protein and mRNA in the cells; plate cloning experiments and Boyden chamber were used to detect cell proliferation and migration in each group cells. In addition, breast cancer xenograft model in nude mice were built with MCF-7 cells transfected BCSG1 siRNA immunohistochemistry and RT-PCR were used to detect the expressions of BCSG1 protein and mRNA in the tissue of nude mice transplanted tumor. Finally, immunohistochemical (SP way) was used to detect the expressions of BCSG1, S100A4, Ets-1, VEGT and CD 105 protein in the tissues of breast cancer and breast benign lesions. In this study, we have investigated the mechanism of invasion and metastasis of breast cancer mechanism in molecular level, which provides a theoretical basis and experimental evidence for targeted therapy of breast cancer.
Materials and methods
1. Effect of BCSG-1 siRNA on the expression of BCSG-1 gene and invasion
of breast cancer MCF-7 cells
Human breast cancer cells MCF-7 were purchased from Kono Bio Co. Ltd. of Nanjing.
MCF-7 cells were cultured in the RPMI-1640 medium containing 100g/L fetal bovine serum, at 37℃in a 5% CO2 humidified atmosphere.
BCSG1 siRNA sequence were designed, synthesized and the synthesis of plasmid vector.
Groups:The normal control group, Empty liposome, Negative control group, BCSG1 siRNA 1 group, BCSG1 siRNA2 group, BCSG1 siRNA3 group, BCSG1 siRNA4 group.
The immunocytochemistry and RT-PCR were used to detect the expression of BCSG1 protein and mRNA.
Plate cloning experiments and Boyden chamber were used to detect proliferation and invasion of MCF-7 cells transfected with the BCSG1 siRNA which inhibits MCF-7 cells according to the results of immunohistochemistry and RT-PCT.
2. Effect of BCSG-1 siRNA on the expression of BCSG-1 gene in tumor tissue of nude mice transplanted with human breast cancer cells
2.1 BALB/C nude mice, female,2 weeks old, were purchased from Hayes Lake Animal Company of limited liability. They were Reared in the conditions of SPF (specific pathogen Free), kept in constant humidity (relative humidity40-60%), constant temperature (26-28℃).
2.2 Groups:The mice were randomly divided into 6 groups,5 each group: group of the mice were inoculated with MCF-7 cells untransfected, group of the mice were inoculated with MCF-7 cells transfected nonsense siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG1 siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG2 siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG3 siRNA sequences, group of the mice were inoculated with MCF-7 cells transfected BCSG4 siRNA sequences.
2.3 Construction of human breast cancer xenograft model in nude mice MCF-7 cells were inoculated subcutaneously into nude mice according to request.Sclerosis, grain of rice, was shown at injection site gradually after inoculating 2 weeks.
2.4 Tumor volume and weight each group were measured.
2.5 Immunohistochemical and RT-PCR were used to detect the expressions of BCSG1 protein and mRNA in nude mice transplanted tumor tissue.
3. The expressions of BCSG1,S100A4,ETS1,VEGF and CD105 protein in human breast cancer tissue.
3.1 Collecting 40 cases of breast fibroadenoma,62 cases of invasive ductal carcinoma, divided into 2 groups according to whether there are lymph node metastasis (respectively 13 and 49 cases), and 48 cases of adjacent, In which 1 group of 13 cases,49 cases of group 2.
3.2 Immunohistochemical was used to detect the expressions of BCSG1, S100A4, Ets1, VEGF and CD 105 protein.
4. Statistical analysis:
Statistical analysis:measurement data were presented as mean value±tandard error of the mean. The differences between two groups were determined with the Student's t test and LSD test. The differences among three or more groups were determined with a one-way ANOVA. Computations were performed using the SPSS 10.0 software. The cutoff for statistical significance was set as P＜0.05.
Results:
1 The changes of the cell morphology
The control group had normal cells morphology. Cells are irregular polygon, and have a clear outline. After BCSG1 siRNA transfection, compared with the control groups, some cells are shrunken, the size become smaller, the cell boundaries become blur, the gap become widened, a small amount of cells are suspended in the culture medium.
2 The immunocytochemistry results
Positive signal of BCGS-1 protein showed brown and located in the cytoplasm. Under the Microscope,10 high power fields were randomly selected, according to the percentage of positive cells and the color depth the results were determined. Rating criteria:(1) the scores according to the percentage of positive:"1%for 0; 1% to 25% for 1 point; 26% to 50%for 2 points; 51% to 75% for 3 points;> 76% to 4 points. (2) the scores according to the color depth of positive cells:no coloring is 0; yellow is 1; brown for 2; tan color is 3. Finally, multiply the final integrated score of two parts. The expressions of BCSG protein in cells of BCSG-1-siRNA-1 group was 4.27±0.12, that of BCSG-1-siRNA-2 group was 4.19±0.22, that of BCSG-1-siRNA-3 group was 4.15±0.14 and that of BCSG-1-siRNA-4 group was 4.17±0.13, independent sequence group was 7.92±0.22, control group was 8.02±0.13, MCF7 control group was 8.16±0.32. Compared with the control groups, the expression of BCSG-1 protein in four groups of specific siRNA transfected cells were decreased, and the differences were statistically significant (P all<0.05), but there was no significant difference four groups of specific siRNA transfection group each other (P>0.05), and there also no significant difference among the control groups, (P> 0.05).
3 Cell RT-PCR results
5μl PCR amplification products and 2μl 6 x Loading Buffer were mixed, spotting in 1%agarose containing ethidium bromide gel, electrophoresis for 20～30min (in 1 x TAE electrophoresis buffer, in 80v voltage; shooting with gel Imaging and recording the scan results; using Quantity One software (BIO-RAD company) to take measurements of the gray value of each band, compared with the calculation of internal referenceβ-actin and got the relative expression level of each gene. BCSG-1 mRNA was all expressed in the MCF-7 cells each group. The relative expression level of BCSG-1 mRNA in BCSG-1-siRNA-1 group was 0.624±0.010, that BCSG-1-siRNA-2 group was 0.626±0.013, BCSG-1-siRNA-3 group was 0.634±0.008, BCSG-1-siRNA-4 group was 0.631±0.010, independent of sequence group was 0.97±0.076, control group was 0.983±0.052, MCF7 control group was 1.014±0.034. Compared with the control groups, the expressions of BCSG-1 mRNA in the groups transfected the four specific siRNA were significantly lower (P all<0.05), and the differences were statistically significant (P all<0.05). There were no statistically significant between the groups transfected four group-specific sequence each other (P all> 0.05) and there no significant difference between the control groups (P> 0.05).
4 Plate cloning experiments results
Single cell suspension of MCF-7 cells were seeded in 60m2 Petri dish, each dish separately with 5×103,10×103 and 20×103 cells. Adding culture medium, gently shaking, the cells dispersed. After 2-3 weeks, terminating cultivation when there is visible cloning cluster. Washed 3 times with PBS solution; Fixed with methanol for 15min, and stained with hematoxylin for 2-3min, Rinsed with running water, dry in air. Colonies including at least 50 cells were counted under microscope. The number of clones 3 dish will be converted into cloning efficiency, and taking the mean. Cloning efficiency = clone number/inoculation cell number×100%. The results showed that:MCF7 control group was 32.33±.52%, empty liposome group was 28.33±3.51%, independent of the control group was 32.00±4.36%, BCSG1 siRNA-3 group was 4.17±0.13%. Compared with the control group, the formation of transfected clones was significantly higher than the control group, and differences were statistically significant (P all<0.05).
5 Boyden chamber experiments
Matrigel glue, diluted with 50μl serum free RPMI-1640 medium, was to uniform evenly blanketed in polycarbonate membrane, irradiated by Uv for 30min when it to become gelatinous after standing for 30min in air 37℃. The cell suspension was diluted to single-cell suspension which concentration was 5×105/ml. According to the requirement, each small room plus 400μl diluted single-cell suspension and each cell plus the three small rooms. They were cultured for 24h under normal conditions; then removing the small room, carefully wiping the film with a cotton swab, fixed with 4% paraformaldehyde,0.1% crystal violet staining, the cells attached to the slide on the side; 5 high power field of view (400×) were randomly selected under microscope, the counting their penetrating cells, Averaging the results. The results show that:penetrating cells in each group, MCF7 control group was 103.53±1.33, empty liposome group was 100.93±1.81, independent of the control group was 101.20±1.71, BCSG1siRNA-3 group was 68.80±10.43. The interference group and control group, the number of penetrating cells significantly decreased, and the difference was statistically significant (P<0.05).
6 The growth of nude mice implanted tumor in each group
The six groups of cells were inoculated into the subcutaneous of the right anterior axillary of nude mice in each group. Scleroma, the size of a grain of rice, gradually appeared at the inoculation site after 3 weeks in the control group, and that appeared in all the groups one week later. And then after 4 weeks, the tumor formation rate in each group is 100%. After the mice were killed, taking out the tumor in each group, weighing, and calculating the rate of tumor growth inhibition (tumor inhibition rate=(control group, tumor weight of a tumor weight of experimental group)/tumor weight of control group x 100%). The results showed:two control groups (A, B) of the tumor mass were 122.34±6.79 and 116.04±5.20, four specific siRNA transfection group (C, D, E, F) the quality of tumor tissue were 20.12±0.85, 21.44±1.43,20.28±0.94 and 20.96±1.42, after the tumor was less than four groups of two control groups, application of statistical methods to compare LSD; four specific siRNA transfection group was no statistical difference between Significance (P> 0.05), the first two control groups no significant difference between (P> 0.05). Compared with the control group, four groups of specific siRNA transfection group nude mice is small and the differences were statistically significant (P＜0.05). The rate of tumor growth inhibition compared to the four specific siRNA transfection group was significantly smaller than the tumor inhibition rate independent of the control group, and the difference was statistically significant (P＜0.05), but four specific siRNA transfection group Tumor inhibition was no significant difference between the rate (P> 0.05).
7 The expression of BCSG1 mRNA in each tumor tissue of nude mice transplanted with MCF-7 cells
The expressions of BCSG1 mRNA in tumor tissue of nude mice transplanted with MCF-7 cells were respectively:BCSG-1-siRNA-1 group was 1.28±0.36, BCSG-1-siRNA-2 group was 1.21±0.38, BCSG-1-siRNA-3 group 1.21±0.47, BCSG-1-siRNA-4 group was 0.86±.17, independent of sequence group 2.19±0.37, control group 2.62±0.68. The implanted tumor in nude mice in unrelated sequence group and blank control group showed obvious BCSG1 mRNA positive band, and that in each BCSG1-siRNA transfected group, only a small amount of mRNA BCSG1 weak positive bands. Compared with each controlled group, the expressions of BCSG1 mRNA were significantly lower (P<0.05). Compared by LSD method, there were no significant difference between groups transfected four specific sequence (P>0.05), the group transfected independent of sequence and the non-transfected control group (P> 0.05).
8 The expression of BCSG1 protein in each tumor tissue of nude mice transplanted wih MCF-7 cells
High-definition image processing system was used to analysis immunohisto chemical image. The results showed:BCSG-1-siRNA-1 group was 8.06±0.21, BCSG-1-siRNA-2 group was 6.98±0.43, BCSG-1-siRNA-3 group was 6.98±0.72, BCSG-1-siRNA-4 group was 7.70＝0.47, independent of sequence group 16.56±0.86, control group 17.90±1.38. Compared with each control group, the expressions of BCSG1 protein were significantly lower (P<0.05),and there were no significant difference between groups transfected four specific sequence (P> 0.05), the group transfected independent of sequence and the non-transfected control group (P> 0.05).
9 The expression of BCSG1 protein in human breast tumor tissue.
The positive rate of BCSG1 protein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma with axillary lymph node metastasis were 2.50%,12.50%, 69.23%, and 77.55%. BCSG1 protein in human breast cancer tissue and normal expression was significantly higher than that of benign (noncancerous) breast tissue, and the differences were statistically significant (P<0.05); the differences in positive rates between benign and malignant tumor were significant (P<0.05); the expression of BCSG1 protein was positively correlated with tumor grade, and increased with the increasing of the degree of tumor invasion and metastasis.
10 The expression of S100A4 protein in human breast tumor tissue.
The positive rate of S100A4 protein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma axillary lymph node metastasis were 5.00%,6.25%, 53.85%,59.18%. Compared with with the benign (noncancerous) breast tissue, the expression of S100A4 protein in human breast cancer tissue and normal expression were significantly higher, and the differences were statistically significant (P<0.05); the differences in positive rates between benign and malignant tumor were significant (p＜0.05).
11 The expression of Etsl protein in human breast tumor tissue.
The positive rate of Etslprotein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma with axillary lymph node metastasis were 12.50%,25.00% and 53.85%,61.22%. The difference in positive rate between the four groups was statistically significant (P<0.05). Compared with the invasive ductal carcinoma with axillary lymph node metastasis, the expression of S100A4 protein in ductal carcinoma without lymph node metastasis no significant changes (P>0.05)
12 The expression of VEGF protein in human breast tumor tissue.
The positive rate of VEGF protein in human breast fibroadenoma, breast tissue adjacent cancer, invasive ductal carcinoma without lymph node metastasis and invasive ductal carcinoma axillary lymph node metastasis were 10.00%,8.33%, 53.85%,73.47%.Compared with the benign (noncancerous) breast tissue, the expression of VEGF protein in human breast cancer tissue and normal expression were significantly higher, and the differences were statistically significant (P <0.05);the differences in positive rates between benign and malignant tumor were significant (P<0.05).
13 The expression of CD105 protein in human breast tumor tissue.
MVD values (X±S) In breast fibroadenoma, ductal carcinoma in situ, invasive ductal carcinoma were 9.33±2.08,18.69±3.37,42.67±14.02. The differences between Benign and malignant was statistically significant (P<0.05), MVD in invasive ductal carcinoma increased significantly.
Conclusion
1. The expression of BCSG1 protein and mRNA in human breast cancer MCF-7 cells and in tumor transplants of human breast cancer cell line in nude mice were high.
2. BCSG1 siRNA can specifically inhibit the expressions of BCSG1 protein and mRNA in the human breast cancer MCF-7 cell line and in the tumor transplants of
human breast cancer cell line in nude mice.
3. BCSG1 siRNA can inhibit breast cancer MCF-7 cells invasive. It guess BCSG1 siRNA treating breast cancer is feasible and provides a new idea for the treatment of breast cancer.
4. BCSG1 siRNA can inhabit the growth of the tumor transplants of human breast cancer cell line in nude mice.
5. The expression of BCSG1 gene is high in human breast cancer.
6. The expression of BCSG1 gene was positively correlated to tumor pathological grade, and with the degree of cancer invasion and metastasis increase, the positive rate of BCSG1 increased.
7. The expression of S100A4、Ets1、VEGF and CD105 protein are all high. BCSG1, S100A4, Ets1, VEGF, CD 105 are closely related to the generate, development and prognosis of breast cancer, and there is great significance on breast cancer prognosis if these five factors are joint detected.
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