乳腺癌EphA2，EphrinA1，ERα，ERβ和PR的表达及RNAi沉默EphA2基因的实验研究
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摘要
乳腺癌是严重危害人类生命健康的常见恶性肿瘤之一,已位居女性肿瘤死亡首位,全球每年约有120余万妇女患乳腺癌,约50万妇女死于乳腺癌。其发病率和死亡率在世界范围内呈较快增长趋势,每年约以0.2～0.3%的速度递增,且发病年龄趋向年轻化；已经证实,年龄、疾病分期、肿瘤大小、淋巴结转移状况和肿瘤微血管密度等临床病理学因素与肿瘤预后密切相关,且早期远处转移和复发是肿瘤致死的主要原因。因此,迫切需要寻找一个敏感、能早期预测乳腺癌复发转移的分子生物学标志物及抑制其复发转移的基因治疗靶点,为临床对乳腺癌的复发和转移的早期预测提供一定的参考依据。
近年来,随着免疫学和分子生物学技术的飞速发展,对乳腺癌标志物其意义的研究也不断增加,从大分子蛋白质标志物到肿瘤基因标志物等达几十种之多,如c-er-B-2,ER, PR, uPA,VEGF, BRCA, muc-1,CD44, maspin, mdr1等,其中对c-erbB-2(HER-2)、ER、PR的研究尤为深入,在乳腺癌的预后评价和治疗中应用最多。但c-erbB-2、ER和PR作为乳腺癌特异性诊断及预测指标,其特异性及灵敏性均不够理想。有必要寻找更好的与预后相关的分子标志物,为临床了解乳腺癌生物学行为、指导治疗提供帮助。
近年来,Eph家族在肿瘤发生和发展过程中的作用受到越来越多的关注,从而为细胞信号传导在肿瘤中的作用机制的深入研究开辟了一个崭新的领域。Eph(erythropoitin producing hepatocellular carcinoma)受体家族是已知最大的受体酪氨酸激酶(receptor tyrosine kinase, RTK)家族亚家族,至今已有16个成员,Eph受体的配体Ephrin (Eph family receptor interacting proteins)已克隆有9个成员。Eph与其配体Ephrin是细胞排斥和粘附的关键调节因子,又是建立、维持及重塑细胞组织形态的基础。功能分析表明,该家族在包括神经网络形成、神经管和轴旁中胚层的成型(patterning)、细胞迁移和神经轴突导向、血管发生等方面具有重要的功能。Eph受体及其配体也与肿瘤发生有关,最近一些基因缺失及体内外血管形成实验表明,Eph和Ephrin基因在各种肿瘤中不同程度的表达与肿瘤的恶性进展有关,如：侵袭力增强,更易于发生转移,血管化程度更明显等,由此而导致患者预后差。
EphA2是Linsberg于1990年从人角化细胞cDNA文库中筛选得到的Eph亚家族成员,是该亚家族成员被发现的具有酪氨酸激酶活性的第一个基因,它在成人上皮细胞中低水平表达,存在于细胞一细胞粘附部位。已证实它在神经元发育、细胞侵袭、黏附调控等方面具有重要作用。与Eph家族其他成员相比,EphA2广泛高表达于不同的肿瘤组织和细胞系,如：乳腺癌、恶性黑色素瘤、前列腺癌和食管癌等。许多证据表明,在肿瘤的血管形成、肿瘤细胞的增殖、侵袭和转移过程中,EphA2发挥着重要作用。已经证实EphA2的高表达能诱导非转化乳腺上皮细胞在体内和体外的恶性转化并促进其侵袭转移。而应用EphA2蛋白的抗体靶向抑制试验,下调EphA2蛋白表达的同时,也抑制了肿瘤细胞的生长,进而降低肿瘤的恶性程度。
EphrinA1是该亚家族第一个被发现的配体,是从肿瘤坏死因子(TNF-a)诱导的人奇静脉内皮细胞中克隆产生。EphrinA 1配体的结合有利于EphA2受体的自身磷酸化。EphrinA1在肿瘤中表达模式的研究刚刚开始,EphA2及EphrinA1共表达于多种肿瘤组织且二者表达明显相关。活化的内生型EphA2与EphrinA1配体相结合,能抑制整合素调节细胞的粘附作用,从而降低细胞粘连,引起高度的组织侵袭性,同时发现EphrinA1配体的密度决定了EphA2介导的整合素调节的细胞粘附作用。这些结果提示EphA2及其配体EphrinA1参与了肿瘤的发生和发展。
新的研究报道EphA2的高表达与许多肿瘤的组织学分级、淋巴结转移及患者的预后有关,在肿瘤治疗方面有潜在的预后价值。但EphA2高表达与各种临床病理学特征的关系在不同类型的肿瘤中表现不一,肺癌组织中高水平的EphA2表达提示患者脑转移的风险增高,但前列腺癌组织却显示EphA2高表达与肿瘤转移无关。目前,国内少见有关EphA2和其配体EphrinA1在乳腺癌中的研究报道。
乳腺癌的发生、发展与人体内雌激素的增加、孕激素的减少密切相关,性激素的代谢素紊乱促进了某些乳腺癌的生长,但其确切发病机制目前尚不清楚。新近在对乳腺癌细胞中EphA2表达状况的研究中,越来越多的证据表明EphA2与ER之间存在相互影响。但是对于EphA2、EphrinA1及ERα、ERβ、PR在乳腺癌中的联合检测,国内外少见报道。
新辅助化疗是对非转移性肿瘤局部治疗之前进行的全身性、系统性细胞毒性药物治疗。目前新辅助化疗已成为局部晚期乳腺癌的标准治疗。国际上关于乳腺癌新辅助化疗的多中心随机临床实验结果显示：新辅助化疗与辅助化疗同样有效。对新辅助化疗敏感的乳腺癌比不敏感者预后好,特别是在局部进展期乳腺癌的应用中已取得良好的效果。新辅助化疗后许多病理学和生物学指标都发生了改变,很多基因表达均发生了改变。寻找出能够预测新辅助化疗疗效并帮助选择内分泌治疗、化疗方案、判断乳腺癌预后的肿瘤分子标记物已经成为目前研究的热点之一,对于EphA2及ER、PR在新辅助化疗前后乳腺癌中的表达的研究,国内外少见报道。
我们通过免疫组织化学技术联合检测了EphA2、EphrinA1及ERα、ERβ、PR的蛋白在乳腺癌中的表达及各自与临床病理学因素的关系；采用免疫组织化学技术检测EphA2和ER, PR在新辅助化疗前后的乳腺癌组织中的表达情况；用RT-PCR技术研究了EphA2及其配体EphrinA1各自的mRNA在乳腺癌中的表达及各自与临床病理学因素的关系；应用逆转录病毒载体介导的RNAi技术抑制EphA2在MCF-7细胞中的表达,以期寻找出能帮助选择有效治疗方案及预测乳腺癌预后的有效指标,并探讨EphA2在乳腺癌中的作用机制及预后价值,以期为乳腺癌的基因治疗提供新的思路。本研究分为四章：
第一章乳腺癌中EphA2,EphrinA1及ERa, ERβ, PR蛋白的表达及其意义
目的
研究EphA2,EphrinA1及ERa, ERP, PR等的蛋白在乳腺癌中的表达及其意义。
方法
1.用免疫组织化学SP法检测130例乳腺癌组织中EphA2, EphrinA1, ERa,ERβ及PR等各自蛋白的表达水平及其与临床病理因素的关系。
2.统计学处理：用spss 13.0 for windows统计软件包对所有资料进行统计学处理。采用卡方检验(chi-square),t(studert t)检验,方差分析(ANVOA),相关性检验等。显著性标准为P<0.05。
结果
1.EphA2,EphrinA1主要位于肿瘤细胞和血管内皮细胞的胞浆中、少量位于炎性细胞的胞浆中,呈棕黄色或棕褐色；ERα、ERβ和PR主要位于细胞核,呈棕黄色或棕褐色,灶性、片状和散在分布,强表达时胞浆也着色。
2.130例乳腺癌组织中,EphA2蛋白阳性表达94例,阳性率72.31%；EphrinA1蛋白阳性表达77例,阳性率59.23%；ERα蛋白阳性表达82例,总阳性率63.08%；ERβ蛋白阳性表达69例,总阳性率53.08%；PR蛋白阳性表达76例,总阳性率58.46%。
3.EphA2蛋白在乳腺癌的阳性表达率与乳腺癌患者的年龄、肿瘤大小和病理类型均无显著相关(P>0.05)；而与临床分期、淋巴结转移和组织学分级显著相关(P<0.05)。临床分期较晚者、淋巴结转移组、组织学分级较高组EphA2蛋白的阳性率分别显著高于临床分期较早者、无淋巴结转移组、组织学分级较低组EphA2蛋白的阳性率。
4.EphrinA1蛋白的阳性表达率与乳腺癌患者的年龄、肿瘤大小、病理类型和淋巴结转移无显著相关(P>0.05)；而与临床分期、组织学分级显著相关(P<0.05),临床分期较晚者和组织学分级较高组EphrinA1蛋白的阳性率分别显著高于临床分期较早者和组织学分级较低组EphrinA1蛋白的阳性率。
5.ERβ和Pβ蛋白的阳性表达率与乳腺癌患者的年龄、肿瘤大小、病理类型、临床分期、淋巴结转移和组织学分级均无显著相关(P>0.05)。
6.ERβ蛋白的阳性表达与乳腺癌患者的年龄、肿瘤大小、病理类型、临床分期和淋巴结转移均无显著相关((P>0.05),ERβ蛋白的阳性表达与组织学分级显著相关(P<0.05),组织学分级较低者ERβ蛋白的阳性率显著低于组织学分级较高者的阳性率。
7.EphA2和EphrinA1蛋白阳性染色共同定位于大致相同的肿瘤区域和血管内皮细胞,二者在乳腺癌组织中的分布基本一致。经统计学分析,二者在乳腺癌组织中的阳性表达显著相关(P<0.05)。
8.EphA2蛋白在乳腺癌中的阳性表达与PR蛋白在乳腺癌中的阳性表达无显著性相关(P>0.05)。EphA2蛋白在乳腺癌中表达的阳性率与ERα蛋白在乳腺癌中的阳性率显著相关(P<0.05),即EphA2蛋白表达的阳性率随着ERα蛋白阳性率的增高呈下降趋势,EphA2蛋白的阳性表达率在乳腺癌ERα蛋白阴性组显著高于ERα蛋白阳性组(P<0.05)。EphA2蛋白在乳腺癌中表达的阳性率与ERβ蛋白在乳腺癌中的阳性率呈显著性相关(P<0.05),即EphA2蛋白表达的阳性率随着ERβ阳性率的增高呈升高趋势。
结论
1.在乳腺癌组织中检测到EphA2、EphrinA1、ERα、ERβ和PR的蛋白的表达,提示他们可能与乳腺癌的发生发展有关。
2.EphA2蛋白在乳腺癌的表达与临床分期、淋巴结转移和组织学分级显著相关,EphrinA1蛋白在乳腺癌的表达与临床分期、组织学分级显著相关,ERβ蛋白在乳腺癌的表达与组织学分级相关,提示其潜在的预后价值并有可能成为乳腺癌预后评估的肿瘤标志物。EphA2蛋白高表达与乳腺癌淋巴结转移有关,提示其可能参与乳腺癌的转移机制。
3.EphA2和EphrinA1在乳腺癌中的蛋白表达分布的一致性提示在乳腺癌中,EphrinA1是EphA2受体的主要配体之一,二者可能在乳腺癌细胞信号传导过程中协调发挥作用。EphA2和EphrinA1共同定位于肿瘤细胞和乳腺癌的血管内皮细胞,提示二者可能协同参与了乳腺癌的血管生成,二者有望成为乳腺癌基因治疗的新靶向。
4.乳腺癌组织中有较高的ERα蛋白和ERβ蛋白阳性表达,提示乳腺癌是性激素依赖性肿瘤,可根据受体的含量指导临床进行内分泌治疗。
5.乳腺癌细胞EphA2与EphrinA1的表达显著相关,提示EphrinA1的表达受EphA2诱导,EphrinA1可以通过自分泌和／或旁分泌环作用于相应的EphA2受体促进癌细胞增殖及血管形成。
6.在乳腺癌中,EphA2蛋白的表达与ERα蛋白负相关,和ERβ蛋白正相关相关,提示ERα可能副性调控EphA2的表达,而ERβ可能正性调控EphA2的表达。
7.EphA2和EphrinA1的检测可作为乳腺癌预后的评估指标。
第二章EphA2、ER、PR在新辅助化疗前后乳腺癌中的表达及意义
目的
研究乳腺癌中ER、PR和EphA2的表达在新辅助化疗前后的变化及临床意义。
方法
采用免疫组化SP法检测ER、PR和EphA2在52例新辅助化疗前后的乳腺癌组织中的表达情况。
结果
ER、PR和EphA2的表达均与疗效相关；新辅助化疗后的乳腺癌组织中EphA2的阳性表达率显著下降(P<0.05);ER、PR的阳性表达率则无明显变化(P>0.05)。
结论
新辅助化疗能抑制肿瘤细胞增殖,促进肿瘤细胞凋亡,显著降低EphA2的阳性表达,而对ER, PR的表达无显著影响。
第三章乳腺癌中EphA2 mRNA和EphrinA1 mRNA的表达及其意义
目的
研究EphA2 mRNA和EphrinA1 mRNA在乳腺癌中的表达及其意义。方法
1.选择40例乳腺癌冰冻标本,分别经免疫组化证实有不同级别的EphA2蛋白表达和EphrinA1蛋白表达,用于RNA提取。
2.应用逆转录-聚合酶链式反应(RT-PCR)分别检测其中EphA2mRNA和EphrinA1mRNA的表达,比较各自mRNA与其蛋白表达的相关性及与临床病理因素的关系。
3.统计学处理：所有数据均经应用SPSS 13.0统计软件包进行统计学处理。应用t检验(t-test);方差分析(One-Way ANVOA);非参数检验(Kruskal-Wallis test法)；显著性标准为P<0.05。
结果
1.乳腺癌组织中EphA2 mRNA的表达与组织学分级、淋巴结转移及临床分期均显著相关(P<0.05)；与患者发病年龄、肿瘤大小、病理类型均无关(P>0.05).
2.乳腺癌组织中EphrinA1 mRNA的表达与组织学分级、淋巴结转移及临床分期均显著相关(P<0.05)；与患者发病年龄、肿瘤大小、病理类型均无关(P>0.05)
3.40例EphA2蛋白表达不同的乳腺癌标本均有EphA2mRNA的表达。10例EphA2蛋白表达0级的mRNA水平(0.34±0.14)明显低于30例EphA2蛋白表达1～3级的mRNA(1级：1.33±0.25；2级：1.55±0.11；3级：1.71±0.19：与0级两两比较：P值均<0.05)水平,在22例EphA2蛋白2～3级表达的样本之间,mRNA表达水平无显著性差异(P>0.05).EphA2蛋白表达和mRNA表达水平不完全一致。
4.40例EphrinA1蛋白表达不同的乳腺癌标本均有EphrinA1 mRNA的表达。7例蛋白0级表达的mRNA水平(0.41±0.11)明显低于33例蛋白表达1～3级的mRNA(1级：1.37±0.38；2级：1.59±0.28；3级：1.67±0.19；与0级两两比较：P值均<0.05)水平,在21例蛋白1～2级表达的样本之间mRNA表达水平无显著性差异(P>0.05),在24例蛋白2～3级表达的样本之间mRNA表达水平也无显著性差异(P>0.05)。EphrinA1蛋白表达和mRNA表达水平不完全一致。
结论
1.乳腺癌癌组织中EphA2 mRNA的表达与组织学分级、淋巴结转移及临床分期均显著相关(P=0.000);EphrinA1 mRNA的表达与组织学分级、淋巴结转移及临床分期相关(P=0.000)；提示EphA2 mRNA的阳性表达和EphrinA 1 mRNA的阳性表达均与乳腺癌的恶性行为相关,有可能成为乳腺癌预后的标记物。
2. EphA2mRNA、EphrinA1mRNA的阳性表达与各自蛋白质的表达具有一定的相关性,提示乳腺癌组织中二者主要在转录水平调节。
3.EphA2 mRNA和EphrinA1 mRNA表达水平分别与各自蛋白表达水平的不完全一致,提示二者在乳腺癌中存在某种转录后翻译水平的调控机制和蛋白质积聚或降解通路障碍。
第四章RNAi靶向沉默乳腺癌细胞系MCF-7中EphA2的表达的研究
目的
探讨逆转录病毒介导的RNAi对乳腺癌细胞系MCF-7中EphA2表达的影响。以期为乳腺癌的基因治疗提供新的思路和手段、
方法
1.根据人EphA2mRNA基因编码区序列,按照序列设计原则及网上软件,设计并合成两条互补的编码EphA2shRNA的寡核苷酸序列。
2.两条互补的编码EphA2shRNA的寡核苷酸链煺火后与线性载体质粒RNAi-READY-pSIREN-RetroQ连接,构建形成重组质粒pSIREN-RetroQ-EphA2,同时用试剂盒提供的阴性对照siRNA双链寡核苷酸构建对照载体pSIREN-RetroQ-C。
3.以重组质粒pSIREN-RetroQ-EphA2转化大肠杆菌。用碱裂解法少量制备质粒DNA,然后进行酶切鉴定和DNA测序分析。
4.脂质体介导下转染到包装病毒细胞株PA317中,并进行克隆与筛选。
5.以重组质粒pSIREN-RetroQ-EphA2转染MCF-7细胞,并用嘌呤霉素筛选稳定表达的克隆。
6.Westen-blot检测EphA2蛋白的表达。
结果
1.重组逆转录病毒载体pSIREN-RetroQ-EphA2经酶切鉴定结果正确；测序分析证实插入的EphA2 siRNA的方向及序列正确。
2.Western blot分析,与对照空载体及未转染细胞相比,重组逆转录病毒表达载体pSIREN-RetroQ-EphA2转染的MCF-7细胞中EphA2蛋白表达水平明显降低。
结论
1.从EphA2 mRNA序列中筛选出有效的siRNA序列,并设计了互补双链寡核苷酸发夹结构,成功构建了EphA2 siRNA的逆转录病毒表达载体,并将之转染到MCF-7细胞。
2.通过逆转录病毒载体介导的RNAi,能够抑制MCF-7细胞中EphA2勺蛋白表达,为观察转染siRNA的逆转录病毒表达载体的乳腺癌细胞恶性生物学行为变化奠定了实验基础,为以EphA2为靶向的乳腺癌的基因治疗提供了新的思路和手段。
Breast carcinoma is one of the most common malignant tumors and has become the number one killer among women cancers.which greatly affects human health and life.It is estimated that 1.2 million of female will be diagnosed of breast carcinoma, with 0.5 million death annually in the world. The incidence and mortality of breast cancer are increasing, with an annual rate of approximately 0.2～0.3%.And more and more young women were affected by this disease.Classical clinical pathological factors such as age, stage of disease, volume, lymphatic spread and microvascular intensity have been confirmed to have a close relationship with prognosis.And disease recurrence and metastasis in early stage are key factors affecting the outcome of initial therapy. Identification of sensitive biomarkers for recurrence and metastatic potential and their regulatory genes in breast cancer cells, therefore, is important for both early clinical detection and effective prophylactic therapy.
As the rapid development of immunological and molecular biological technologies in the past few decades, the clinical and pathological significance of many biomarkers such as c-er-B-2,ER, PR, uPA,VEGF, BRCA, muc-1,CD44, maspin, mdrl,etc,have been studied in breast cancer at cellular and molecular levels. Among all these biomarkers, c-erbB-2(HER-2), ER and PR are widely studied and considered to be of important prognostic values.But using c-erbB-2,ER and PR as diagnosis and prognostic markers are still not satisfactory enough. However, up to now, it is still mandatory to find better survival prognostic markers to better understand the biological behavior of breast carcinoma, supply with better clinical management of breast carcinoma.
Recently, more and more investigators have focused on the roles of Eph family in tumours, which extends a new wide area for deep researches of the potential mechanisms of signal transduction in carcinomas.The Eph (erythropoitin producing hepatocellular carcinoma) receptor family is the largest subfamily of receptor protein tyrosine kinases (RTK),consisting of at least sixteen distinct receptors and nine membrane-bound ligands, known as Ephrins(Eph family receptor interacting proteins).Signal conduction mediated by Eph receptors and their ligands Ephrins is important to cell-cell repulsion and adhesion, which is also the base of the formation, patterning and plasticity of tissue. Functional studies have demonstrated that Eph receptors and ephrins play important roles in many developmental processes including formation of neuronal network, patterning of neural tube and paraxial mesoderm, guidance of cell migration and axon pathfinding, and vascular formation. Recent studies have also suggested that Eph receptors and ephrins may be involved in carcinogenesis.Recently, gene targeting experiments and angiogenesis assay in vitro and vivo indicates that the genes for Eph receptors and ephrins have been recognized to be differentially expressed in various human tumors.The aberrant expression is associatied with the altered tumor behavior, such as increased invasiveness, increased metas;tatic potential,prominent vascularization, and thus a poor prognosis.
EphA2 was a number of Eph subfamily sifted from human horny cDNA library by Linsberg in 1990,it was the first gene discovered with tyrosine kinase active in Eph subfamily. EphA2 is a transmembrane receptor tyrosine kinase that is found at low levels on adult epithelial cells and enriched within sites of cell-cell adhesion and has been implicated in neuronal development, as well as regulating cell migration and adhesion.Compared with other members of Eph family, EphA2 has been shown frequently overexpressed in multiple types of carcinoma tissues and cell lines, such as, mammary cancer, malignant melanoma, prostatic carcinoma and esphageal carcinomas, et al.Many investigations suggest that EphA2 has a important role in the regulation of angiopoiesis, cell proliferation, invasion and metastasis of carcinoma. It has been proved that the high level of EphA2 can induce the maglignant transformation of mammary epithelial cells in vitro and vivo and it can also promote the invasion and metastasis of the carcinoma. Interestingly, the test of targeting inhibition of EphA2 protein in vitro have shown that using anti-EphA2 antibody can down-regulates expression of EphA2 and inhibits tumour cell growth, and thus lower the malignant degree of the tumor.
EphrinAl (B16) is the major and also the first ligand discovered for EphA2 and was originally cloned from human umbilical vein endothelial cells as a factor induced by tumor necrosis factor-a(TNF-α).Ligand binding contributes to EphA2 autophosphorylation. However, little is known about its expression pattern in tumours. EphA2 and EphrinAl co-overexpressed in many malignant carcinomas and it is reported that the expression of EphA2 correlates with EphrinAl expression. Some study have reported that activation of endogenous EphA2 kinase stimulated by EphrinAl ligand can suppresse integrin-regulated cell adhesion, decreasing cell-cell attachment, endows tumour cells with more aggressive ability. Meanwhile,they found that cell adhesion mediated by integrin is mainly determined by density of EphrinAl ligand. All of these studies indicated that EphA2 and EphrinAl may be involved in the process of tumorigenesis and development of tumours.Recent research reported that the overexpression of EphA2 is related to histological grade, lymph node metastases, and poor prognosis, Suggesting its potential prognostic value in tumor treatment. But recent investigations have indicated that the relationships between overexpression of EphA2 and all sorts of clinicopathological features were different in different typies of tumours.For example, high levels of EphA2 protein in lung cancer predict a high risk of brain metastasis of patients, however, high levels of EphA2 protein did not relate to metastasis in prostatic carcinomas.However, few reports concerned with the expressions of EphA2 and EphrinAl in breast carcinoma in literature in our country is available.
Known and suspected causes for oncogenesis and development of breast cancer include high estrogen level and low progestin level.Metabolic disorder of steroid hormone promotes the development of some kinds of breast cancer, but the involved regulation mechanism remains as yet unclear. Evidence is accumulating that the changes of EphA2 in breast carcinoma interact with ER. But there is little literature about the combined detection of the protein of EphA2,EphrinAl,ERα, ERβand PR in breast carcinoma.
Neo-adjuvant chemotherapy is a method of therapy for non-metastatic cancer that involves systemic treatment with the application of cytotoxic drugs before the local treatment.Currently, the new adjuvant chemotherapy has become the standard treatment for locally advanced breast cancer. The results of the international multi-center randomized clinical trial on neo-adjuvant chemotherapy for breast cancer showed that:Neo-adjuvant chemotherapy is as useful as adjuvant chemotherapy for malignant tumors.The patients with breast cancer that are sensitive to neo-adjuvant chemotherapy have a better prognosis than those who are not sensitive to it. And, good results with the application of the therapy in patients with locally advanced breast carcinoma have already been obtained.With the neo-adjuvant chemotherapy treatment, many pathological and biological indicators of breast carcinoma have changed, and the expression of many genes were altered too.To find out the molecular markers which can predict the efficacy of neo-adjuvant chemotherapy and help to select effective endocrine therapeutic regimen, chemotherapy plan, and to determine the prognosis of breast carcinoma has become a hot spot in the current study. While recent researches on expression of EphA2 and ER, PR in breast carcinoma before and after neoadjuvant chemotherapy is few overseas, and less in China.
Using immunohistochemistry technology to detect the protein expression of EphA2, EphrinAl,ERα, ERβand PR in breast carcinomas, and then studying their relation to clinicopathological factors;The expressions of ER、PR and EphA2 were detected by immunohistochemical method in breast cancer tissues before and after Neo-adjuvant Chemotherapy. We systematically analyzed the expression patterns of EphA2 and EphrinAl in a series of breast carcinomas by RT-PCR technology, explored the relationship between mRNA expressions of EphA2 and EphrinAl and clinicopathological features;In addition, suppression of the expression of EphA2 in MCF-7 cells by using retrovirus-mediated RNAi method was also conducted, In order to find the effective indicators that can help to select effective strategy for treatment and predict prognosis, and to explore the mechanism and prognostic value of EphA2 in breast carcinomas, and to provide new ideas and theoretical basis for gene therapy for breast cancer.This study is divided into four parts as listed below:
Part One:Protein Expressions of EphA2, EphrinA1, ERα, ERβ, and PR in Breast Carcinomas and Their Significance
Objectives:
To study the protein expressions of EphA2,EphrinAl, ERα, ERβ,and PR in breast carcinomas and to explore their significance.
Methods
1.Using Immunohistochemistry Sp method to detect the protein expressions of EphA2, EphrinAl, ERα, ERβ, and PR in tumors from 130 breast carcinomas. Then studying their relationship to clinicopathological factors.
2.The SPSS version 13.0 Statistical package was used for the statistical analysis. We also usedχ2-test(chi-square), t-test(studentt), ANVOA method, and other correlated tests.values of<0.05 were considered statistically significant.
Results
1.The distribution of EphA2,EphrinAl,ERa, ERP and PR was identical, EphA2 and EphrinAl are mainly located in the cytoplasm of tumor cells and endothelial cells and less in the cytoplasm of inflammatory cells. Positive expressions of ERα, ERβand PR were observed in the nucleus of the tumor cells.
2.Among the 130 breast carcinoma cases,94 cases for EphA2 protein were positive,total positive rate was 72.31%;For EphrinAl protein expression,77 cases were positive, total positive rate was 59.23%;For ERαprotein expression,82 cases were positive, total positive rate was 63.08%;For ERβprotein expression,69 cases were positive, total positive rate was 53.08%;67 cases for PR protein expression were positive,total positive rate was 53.08%.
3.The positive expression of EphA2 protein were not associated with age, tumor size and pathological type(P>0.05);But with clinical stage,lymph node metastasis and histological grade(P<0.05).The expression of EphA2 protein of the group with late clinical stage,lymph node metastasis, and high histological grade was respectively higher than the group with early clinical stage, negative lymph node metastasis and low histological grade.
4.The positive expression of EphrinAl protein were not related to age, tumor size, pathological type and lymph node metastasis(P>0.05),but significantly correlated with clinical stage and histological grade(P<0.05).The expression of EphrinA1 protein of the group with late clinical stage, high histological grade was respectively higher than the group with early clinical stage and low histological grade.
5.The protein expressions of ERa and PR were not associated with age,tumor size,pathological type, clinical stage, lymph node metastasis and histological grade(P >0.05).
6.The positive expression of ERβprotein were not associated with age, tumor size, pathological type, clinical stage,and lymph node metastasis(P>0.05), but with histological grade(P<0.05).The expression of ERβprotein of the group with high histological grade was higher than the group with low histological grade.
7.The positive staining of EphA2 and EphrinAl protein co-located in roughly the same tumor areas and vascular endothelial cells.Their distribution are fundamentally unanimous and their expression were positively correlated (P<0.05).
8.Their were no significant correlations between the positive expression of EphA2 protein and the positive expression of PR protein(P>0.05).The exprssion of EphA2 protein was significantly correlated with the expression of ER in breast carcinoma(P<0.05),the positive rate of EphA2 protein expression decreased when the positive rate of ERa protein increased.The positive rate of EphA2 protein in ERa protein-negative group was significantly higher than ERa protein-positive group.The exprssion of EphA2 protein was significantly correlated with the expression of ERβin breast carcinoma(P<0.05),the positive rate of EphA2 protein expression increased when the positive rate of ERβprotein increased.
Conclusions
1.Through the study, we found that EphA2,EphrinA1,ERα, ERβand PR expressed in breast cancer, it is indicated that they may be involved in carcinogenesis, development procedures of breast carcinoma.
2.The positive expression of EphA2 protein were associated with clinical stage, lymph node metastasis and histological grade.The protein expression of EphrinA1 were related to clinical stage and histological grade.The positive expression of ERβprotein were associated with histological grade.These indicated that they may have important potential prognosis value and may be used as new markers to the prognosis evaluation of breast carcinoma. The overexpression of EphA2 is associated with lymph node metastases, indicating the possibility that it may be involved in metastasis of breast carcinoma.
3.Consistent pattern of protein expressions of EphA2 and EphrinAl in breast carcinoma reveals EphrinAl is one of the major ligands which interacted with EphA2 receptor, and EphA2 and EphrinAl could coordinate with each other in signal conduction in breast carcinoma. EphA2 and EphrinAl colocalized in tumour cells and vascular endothelial cells in breast carcinomas, suggesting that EphA2 and EphrinAl may may be invovled in tumour angiogenesis, and therefore could be attractive new targets for breast cancer therapy.
4.Protein expression of ERP and ERa in breast carcinomas suggesting that breast cancer is a kind of hormone-dependent tumor, and we can give directions to clinical endocrine treatment in accordance with the receptor content.
5.There were significantly correlations between EphA2 and EphrinA1,and hinted the expression of EphrinAl was induced by EphA2,EphrinA1 acted on its receptor EphA2 in an autocrine and/or paracrine manner to promote the growth of tumor cells and proliferation of endothelial cells.
6.The protein exprssion of EphA2 were significantly negatively correlated with the expression of ERa protein, and positively correlated with ERβprotein in breast carcinoma(all P<0.05),It old us that EphA2 may be negative regulated by ERa, and may be positively regulated by ERβ.
7. Detecting of the expression of EphA2 and EphrinA1 may be valuable for evaluating the prognosis of breast carcinoma.
Part two:Expressions of ER、PR and EphA2 in Breast Carcinomas with Neoadjuvant Chemotherapy and Their Significance
Objectives
To study the influence of neo-adjuvant chemotherapy on expression of ER、PR and EphA2 in breast cancer and their clinical sigficance.
Methods
The expressions of ER、PR and EphA2 were detected by SP immunohistochemical method in breast cancer tissues of 52 patients before and after Neoadjuvant Chemotherapy.
Results
The expressions of ER、PR and EphA2 were significantly related to treatment response(P<0.05).The positive rate of EphA2 in breast cancer tissues after neoadjuvant chemotherapy was significantly lower than that before the treatment(P<0.05),but The difference of ER、PR before and after Neoadjuvant chemotherapy were insignificant(P>0.05).
Conclusions
Neoadjuvant chemotherapy could inhibit tumor proliferation and induce tumor cell apoptosis,and significantly decrease the expression of EphA2,but it could not reduce the expression of ER and PR significantly.
Part Three:The expressions of EphA2 mRNA and EphrinAl mRNA in breast carcinomas and their significance
Objectives:
To study the expressions of EphA2 mRNA and EphrinAl mRNA in breast carcinoma and their significance
Methods
1.Frozen tissues from 40 cases in which the tumours showed different grades of EphA2 and EphrinAl protein expressions were used for extraction of mRNA.
2.The levels of EphA2 and EphrinA 1 mRNA were detected by RT-PCR and compared if the levels of mRNA correspond to their levels of protein and clinicopathological features.
3.Statistical analysis:All the data were analyzed by the SPSS version 13.0 statistical package.We have used t-test(student t),One-way ANVOA and Nonparametric test(Kruskal-Wallis test method).P values of<0.05 were considered statistically significant.
Results:
1.The positive expressions of EphA2 mRNA were associated with histological grade,lymph node metastasis and clinical stage(P<0.05),But not with age,tumor size and pathological type(P>0.05).
2.The positive expressions of EphrinAl mRNA were associated with histological grade, lymph node metastasis and clinical stage(P<0.05),But not with age,tumor size and pathological type(P>0.05).
3.All the 40 breast carcinoma specimens with different grade EphA2 protein expression have expressed EphA2 mRNA.The mRNA expression levels in those 10 cases with negative protein expression (0.34±0.14) were obviously lower than those 30 cases with 1～3 grade protein expression(protein gradel:1.33±0.25,grade 2: 1.55±0.11,grade 3:1.71±0.19,each compare with grade 0:all P<0.05).Among the 22 cases with protein expression form grade 2 to 3,There were no significant difference in their mRNA expression levels(P>005).Indicating that EphA2mRNA expression did not fully correspond to its protein expression in breast carcinoma.
4. All the 40 breast carcinoma specimens with different grade EphrinAl protein expression have expressed EphrinAl mRNA.The mRNA expression levels in those 7 cases with negative protein expression(0.41±0.11)were obviously lower than those 33 cases with 1～3 grade protein expression(protein gradel:1.37±0.38, grade 2: 1.59±0.28,grade 3:1.67±0.19, each compare with grade 0:all P<0.05).Among the 21 cases with protein expression form grade 1 to 2,There were no significant difference in their mRNA expression levels(P>005).Among the 24 cases with protein expression form grade 2 to 3,There were no significant difference in their mRNA expression levels(P>005).Indicating that EphrinA1 mRNA expression did not fully correspond to its protein expression in breast carcinoma.
Conclusions
1.The positive expressions of EphA2 mRNA and EphrinAl mRNA in breast carcinoma were associated with histological grade, lymph node metastasis and clinical stage(P<0.05).This indicated that the positive expressions of EphA2 mRNA and EphrinAl mRNA were related to the malignant biological properties of breast carcinoma. And indicating that they may become new markers to the evaluation of the prognosis of breast carcinoma.
2.To some extent, the positive expression of EphA2mRNA and EphrinA1 mRNA in breast carcinoma were associated with the expression of EphA2 protein and EphrinAl protein respectively, indicating that they were both regulated at transcriptional level.
3.The positive expression of EphA2mRNA and EphrinAlmRNA in breast carcinoma were partly different from the expression of EphA2 protein and EphrinAl protein respectively, indicating that there may be some post-transcriptional and translational regulation mechanisms, some accumulation obstacles and some degradation pathway disorders.
Part four:Study of targeted silencing of EphA2 in breast cancer cell line MCF-7 by RNA interference
Objectives
To investigate the effects of retrovirus-mediated siRNA targeting EphA2 on the breast cancer cell line MCF-7, in order to provide the possibility of theoretical basis of gene therapy in vitro for breast cancer.
Methods
1.Based on the human EphA2mRNA gene sequencing and according to the design principle, two complementary oligodeoxyribonucleotides encoding EphA2 short hairpin RNA were designed and synthesized with the software online.
2.Then these two complementary oligodeoxyribonucleotides were annealed and ligated into a linear vector RNAi-READY-pSIREN-RetroQ to construct the recombinant plasmid pSIREN-RetroQ-EphA2.The control vector (pSIREN-RetroQ-C) was constructed at the same time which contain a free correlation sequence.
3.The ligation mixtures were transformed into competent E.coli DH5a. And the recombinant plasmids pSIREN-RetroQ-EphA2 were extracted from small-scale bacterial cultures by Alkaline Lysis, Subsequently the plasmids were digested and then identified by DNA sequence analysis.
4.The vectors based RNAi were transfected into packing cell line PA317,which was selected by puromycin later.
5.MCF-7 cells were transfected by recombinant plasmids pSIREN-RetroQ-EphA2 and stable transfectants were isolated by puromycin.
6.The EphA2 protein in transfected MCF-7 cells was detected by Western blots.
Results
1.The pSIREN-RetroQ-EphA2 vector was confirmed by restriction endonuclease digestion and It was verified by DNA sequencing that the insert sequence was successfully cloned into the vector.
2.Compared with cells transfected with empty retroviru vector and untransfected cells, the level of EphA2 protein expression was greatly decreased in MCF-7 cells transfected by the recombinant retroviru vector by Western blots techniques.
Conclusions
1.We selected effective EphA2 siRNA sequence from EphA2 mRNA sequence and designed and synthesized the hairpin structure of double complementary chains of oligonucleotides,The recombinant retro viral vectors were constructed successfully and retroviru has been successfully transfected into MCF-7 cells line and it works well.
2.EphA2 protein expression in MCF-7 cell line is successfully suppressed by using the recombinant retroviru-mediated RNAi technique, indicating that this may contributes to the study of the changes of malignant biological activity of breast cancer cell lines by transfection of siRNA retrviral vectors and provide a new solution for EphA2-targeting therapeutic intervention of breast carcinoma.
近年来,随着免疫学和分子生物学技术的飞速发展,对乳腺癌标志物其意义的研究也不断增加,从大分子蛋白质标志物到肿瘤基因标志物等达几十种之多,如c-er-B-2,ER, PR, uPA,VEGF, BRCA, muc-1,CD44, maspin, mdr1等,其中对c-erbB-2(HER-2)、ER、PR的研究尤为深入,在乳腺癌的预后评价和治疗中应用最多。但c-erbB-2、ER和PR作为乳腺癌特异性诊断及预测指标,其特异性及灵敏性均不够理想。有必要寻找更好的与预后相关的分子标志物,为临床了解乳腺癌生物学行为、指导治疗提供帮助。
近年来,Eph家族在肿瘤发生和发展过程中的作用受到越来越多的关注,从而为细胞信号传导在肿瘤中的作用机制的深入研究开辟了一个崭新的领域。Eph(erythropoitin producing hepatocellular carcinoma)受体家族是已知最大的受体酪氨酸激酶(receptor tyrosine kinase, RTK)家族亚家族,至今已有16个成员,Eph受体的配体Ephrin (Eph family receptor interacting proteins)已克隆有9个成员。Eph与其配体Ephrin是细胞排斥和粘附的关键调节因子,又是建立、维持及重塑细胞组织形态的基础。功能分析表明,该家族在包括神经网络形成、神经管和轴旁中胚层的成型(patterning)、细胞迁移和神经轴突导向、血管发生等方面具有重要的功能。Eph受体及其配体也与肿瘤发生有关,最近一些基因缺失及体内外血管形成实验表明,Eph和Ephrin基因在各种肿瘤中不同程度的表达与肿瘤的恶性进展有关,如：侵袭力增强,更易于发生转移,血管化程度更明显等,由此而导致患者预后差。
EphA2是Linsberg于1990年从人角化细胞cDNA文库中筛选得到的Eph亚家族成员,是该亚家族成员被发现的具有酪氨酸激酶活性的第一个基因,它在成人上皮细胞中低水平表达,存在于细胞一细胞粘附部位。已证实它在神经元发育、细胞侵袭、黏附调控等方面具有重要作用。与Eph家族其他成员相比,EphA2广泛高表达于不同的肿瘤组织和细胞系,如：乳腺癌、恶性黑色素瘤、前列腺癌和食管癌等。许多证据表明,在肿瘤的血管形成、肿瘤细胞的增殖、侵袭和转移过程中,EphA2发挥着重要作用。已经证实EphA2的高表达能诱导非转化乳腺上皮细胞在体内和体外的恶性转化并促进其侵袭转移。而应用EphA2蛋白的抗体靶向抑制试验,下调EphA2蛋白表达的同时,也抑制了肿瘤细胞的生长,进而降低肿瘤的恶性程度。
EphrinA1是该亚家族第一个被发现的配体,是从肿瘤坏死因子(TNF-a)诱导的人奇静脉内皮细胞中克隆产生。EphrinA 1配体的结合有利于EphA2受体的自身磷酸化。EphrinA1在肿瘤中表达模式的研究刚刚开始,EphA2及EphrinA1共表达于多种肿瘤组织且二者表达明显相关。活化的内生型EphA2与EphrinA1配体相结合,能抑制整合素调节细胞的粘附作用,从而降低细胞粘连,引起高度的组织侵袭性,同时发现EphrinA1配体的密度决定了EphA2介导的整合素调节的细胞粘附作用。这些结果提示EphA2及其配体EphrinA1参与了肿瘤的发生和发展。
新的研究报道EphA2的高表达与许多肿瘤的组织学分级、淋巴结转移及患者的预后有关,在肿瘤治疗方面有潜在的预后价值。但EphA2高表达与各种临床病理学特征的关系在不同类型的肿瘤中表现不一,肺癌组织中高水平的EphA2表达提示患者脑转移的风险增高,但前列腺癌组织却显示EphA2高表达与肿瘤转移无关。目前,国内少见有关EphA2和其配体EphrinA1在乳腺癌中的研究报道。
乳腺癌的发生、发展与人体内雌激素的增加、孕激素的减少密切相关,性激素的代谢素紊乱促进了某些乳腺癌的生长,但其确切发病机制目前尚不清楚。新近在对乳腺癌细胞中EphA2表达状况的研究中,越来越多的证据表明EphA2与ER之间存在相互影响。但是对于EphA2、EphrinA1及ERα、ERβ、PR在乳腺癌中的联合检测,国内外少见报道。
新辅助化疗是对非转移性肿瘤局部治疗之前进行的全身性、系统性细胞毒性药物治疗。目前新辅助化疗已成为局部晚期乳腺癌的标准治疗。国际上关于乳腺癌新辅助化疗的多中心随机临床实验结果显示：新辅助化疗与辅助化疗同样有效。对新辅助化疗敏感的乳腺癌比不敏感者预后好,特别是在局部进展期乳腺癌的应用中已取得良好的效果。新辅助化疗后许多病理学和生物学指标都发生了改变,很多基因表达均发生了改变。寻找出能够预测新辅助化疗疗效并帮助选择内分泌治疗、化疗方案、判断乳腺癌预后的肿瘤分子标记物已经成为目前研究的热点之一,对于EphA2及ER、PR在新辅助化疗前后乳腺癌中的表达的研究,国内外少见报道。
我们通过免疫组织化学技术联合检测了EphA2、EphrinA1及ERα、ERβ、PR的蛋白在乳腺癌中的表达及各自与临床病理学因素的关系；采用免疫组织化学技术检测EphA2和ER, PR在新辅助化疗前后的乳腺癌组织中的表达情况；用RT-PCR技术研究了EphA2及其配体EphrinA1各自的mRNA在乳腺癌中的表达及各自与临床病理学因素的关系；应用逆转录病毒载体介导的RNAi技术抑制EphA2在MCF-7细胞中的表达,以期寻找出能帮助选择有效治疗方案及预测乳腺癌预后的有效指标,并探讨EphA2在乳腺癌中的作用机制及预后价值,以期为乳腺癌的基因治疗提供新的思路。本研究分为四章：
第一章乳腺癌中EphA2,EphrinA1及ERa, ERβ, PR蛋白的表达及其意义
目的
研究EphA2,EphrinA1及ERa, ERP, PR等的蛋白在乳腺癌中的表达及其意义。
方法
1.用免疫组织化学SP法检测130例乳腺癌组织中EphA2, EphrinA1, ERa,ERβ及PR等各自蛋白的表达水平及其与临床病理因素的关系。
2.统计学处理：用spss 13.0 for windows统计软件包对所有资料进行统计学处理。采用卡方检验(chi-square),t(studert t)检验,方差分析(ANVOA),相关性检验等。显著性标准为P<0.05。
结果
1.EphA2,EphrinA1主要位于肿瘤细胞和血管内皮细胞的胞浆中、少量位于炎性细胞的胞浆中,呈棕黄色或棕褐色；ERα、ERβ和PR主要位于细胞核,呈棕黄色或棕褐色,灶性、片状和散在分布,强表达时胞浆也着色。
2.130例乳腺癌组织中,EphA2蛋白阳性表达94例,阳性率72.31%；EphrinA1蛋白阳性表达77例,阳性率59.23%；ERα蛋白阳性表达82例,总阳性率63.08%；ERβ蛋白阳性表达69例,总阳性率53.08%；PR蛋白阳性表达76例,总阳性率58.46%。
3.EphA2蛋白在乳腺癌的阳性表达率与乳腺癌患者的年龄、肿瘤大小和病理类型均无显著相关(P>0.05)；而与临床分期、淋巴结转移和组织学分级显著相关(P<0.05)。临床分期较晚者、淋巴结转移组、组织学分级较高组EphA2蛋白的阳性率分别显著高于临床分期较早者、无淋巴结转移组、组织学分级较低组EphA2蛋白的阳性率。
4.EphrinA1蛋白的阳性表达率与乳腺癌患者的年龄、肿瘤大小、病理类型和淋巴结转移无显著相关(P>0.05)；而与临床分期、组织学分级显著相关(P<0.05),临床分期较晚者和组织学分级较高组EphrinA1蛋白的阳性率分别显著高于临床分期较早者和组织学分级较低组EphrinA1蛋白的阳性率。
5.ERβ和Pβ蛋白的阳性表达率与乳腺癌患者的年龄、肿瘤大小、病理类型、临床分期、淋巴结转移和组织学分级均无显著相关(P>0.05)。
6.ERβ蛋白的阳性表达与乳腺癌患者的年龄、肿瘤大小、病理类型、临床分期和淋巴结转移均无显著相关((P>0.05),ERβ蛋白的阳性表达与组织学分级显著相关(P<0.05),组织学分级较低者ERβ蛋白的阳性率显著低于组织学分级较高者的阳性率。
7.EphA2和EphrinA1蛋白阳性染色共同定位于大致相同的肿瘤区域和血管内皮细胞,二者在乳腺癌组织中的分布基本一致。经统计学分析,二者在乳腺癌组织中的阳性表达显著相关(P<0.05)。
8.EphA2蛋白在乳腺癌中的阳性表达与PR蛋白在乳腺癌中的阳性表达无显著性相关(P>0.05)。EphA2蛋白在乳腺癌中表达的阳性率与ERα蛋白在乳腺癌中的阳性率显著相关(P<0.05),即EphA2蛋白表达的阳性率随着ERα蛋白阳性率的增高呈下降趋势,EphA2蛋白的阳性表达率在乳腺癌ERα蛋白阴性组显著高于ERα蛋白阳性组(P<0.05)。EphA2蛋白在乳腺癌中表达的阳性率与ERβ蛋白在乳腺癌中的阳性率呈显著性相关(P<0.05),即EphA2蛋白表达的阳性率随着ERβ阳性率的增高呈升高趋势。
结论
1.在乳腺癌组织中检测到EphA2、EphrinA1、ERα、ERβ和PR的蛋白的表达,提示他们可能与乳腺癌的发生发展有关。
2.EphA2蛋白在乳腺癌的表达与临床分期、淋巴结转移和组织学分级显著相关,EphrinA1蛋白在乳腺癌的表达与临床分期、组织学分级显著相关,ERβ蛋白在乳腺癌的表达与组织学分级相关,提示其潜在的预后价值并有可能成为乳腺癌预后评估的肿瘤标志物。EphA2蛋白高表达与乳腺癌淋巴结转移有关,提示其可能参与乳腺癌的转移机制。
3.EphA2和EphrinA1在乳腺癌中的蛋白表达分布的一致性提示在乳腺癌中,EphrinA1是EphA2受体的主要配体之一,二者可能在乳腺癌细胞信号传导过程中协调发挥作用。EphA2和EphrinA1共同定位于肿瘤细胞和乳腺癌的血管内皮细胞,提示二者可能协同参与了乳腺癌的血管生成,二者有望成为乳腺癌基因治疗的新靶向。
4.乳腺癌组织中有较高的ERα蛋白和ERβ蛋白阳性表达,提示乳腺癌是性激素依赖性肿瘤,可根据受体的含量指导临床进行内分泌治疗。
5.乳腺癌细胞EphA2与EphrinA1的表达显著相关,提示EphrinA1的表达受EphA2诱导,EphrinA1可以通过自分泌和／或旁分泌环作用于相应的EphA2受体促进癌细胞增殖及血管形成。
6.在乳腺癌中,EphA2蛋白的表达与ERα蛋白负相关,和ERβ蛋白正相关相关,提示ERα可能副性调控EphA2的表达,而ERβ可能正性调控EphA2的表达。
7.EphA2和EphrinA1的检测可作为乳腺癌预后的评估指标。
第二章EphA2、ER、PR在新辅助化疗前后乳腺癌中的表达及意义
目的
研究乳腺癌中ER、PR和EphA2的表达在新辅助化疗前后的变化及临床意义。
方法
采用免疫组化SP法检测ER、PR和EphA2在52例新辅助化疗前后的乳腺癌组织中的表达情况。
结果
ER、PR和EphA2的表达均与疗效相关；新辅助化疗后的乳腺癌组织中EphA2的阳性表达率显著下降(P<0.05);ER、PR的阳性表达率则无明显变化(P>0.05)。
结论
新辅助化疗能抑制肿瘤细胞增殖,促进肿瘤细胞凋亡,显著降低EphA2的阳性表达,而对ER, PR的表达无显著影响。
第三章乳腺癌中EphA2 mRNA和EphrinA1 mRNA的表达及其意义
目的
研究EphA2 mRNA和EphrinA1 mRNA在乳腺癌中的表达及其意义。方法
1.选择40例乳腺癌冰冻标本,分别经免疫组化证实有不同级别的EphA2蛋白表达和EphrinA1蛋白表达,用于RNA提取。
2.应用逆转录-聚合酶链式反应(RT-PCR)分别检测其中EphA2mRNA和EphrinA1mRNA的表达,比较各自mRNA与其蛋白表达的相关性及与临床病理因素的关系。
3.统计学处理：所有数据均经应用SPSS 13.0统计软件包进行统计学处理。应用t检验(t-test);方差分析(One-Way ANVOA);非参数检验(Kruskal-Wallis test法)；显著性标准为P<0.05。
结果
1.乳腺癌组织中EphA2 mRNA的表达与组织学分级、淋巴结转移及临床分期均显著相关(P<0.05)；与患者发病年龄、肿瘤大小、病理类型均无关(P>0.05).
2.乳腺癌组织中EphrinA1 mRNA的表达与组织学分级、淋巴结转移及临床分期均显著相关(P<0.05)；与患者发病年龄、肿瘤大小、病理类型均无关(P>0.05)
3.40例EphA2蛋白表达不同的乳腺癌标本均有EphA2mRNA的表达。10例EphA2蛋白表达0级的mRNA水平(0.34±0.14)明显低于30例EphA2蛋白表达1～3级的mRNA(1级：1.33±0.25；2级：1.55±0.11；3级：1.71±0.19：与0级两两比较：P值均<0.05)水平,在22例EphA2蛋白2～3级表达的样本之间,mRNA表达水平无显著性差异(P>0.05).EphA2蛋白表达和mRNA表达水平不完全一致。
4.40例EphrinA1蛋白表达不同的乳腺癌标本均有EphrinA1 mRNA的表达。7例蛋白0级表达的mRNA水平(0.41±0.11)明显低于33例蛋白表达1～3级的mRNA(1级：1.37±0.38；2级：1.59±0.28；3级：1.67±0.19；与0级两两比较：P值均<0.05)水平,在21例蛋白1～2级表达的样本之间mRNA表达水平无显著性差异(P>0.05),在24例蛋白2～3级表达的样本之间mRNA表达水平也无显著性差异(P>0.05)。EphrinA1蛋白表达和mRNA表达水平不完全一致。
结论
1.乳腺癌癌组织中EphA2 mRNA的表达与组织学分级、淋巴结转移及临床分期均显著相关(P=0.000);EphrinA1 mRNA的表达与组织学分级、淋巴结转移及临床分期相关(P=0.000)；提示EphA2 mRNA的阳性表达和EphrinA 1 mRNA的阳性表达均与乳腺癌的恶性行为相关,有可能成为乳腺癌预后的标记物。
2. EphA2mRNA、EphrinA1mRNA的阳性表达与各自蛋白质的表达具有一定的相关性,提示乳腺癌组织中二者主要在转录水平调节。
3.EphA2 mRNA和EphrinA1 mRNA表达水平分别与各自蛋白表达水平的不完全一致,提示二者在乳腺癌中存在某种转录后翻译水平的调控机制和蛋白质积聚或降解通路障碍。
第四章RNAi靶向沉默乳腺癌细胞系MCF-7中EphA2的表达的研究
目的
探讨逆转录病毒介导的RNAi对乳腺癌细胞系MCF-7中EphA2表达的影响。以期为乳腺癌的基因治疗提供新的思路和手段、
方法
1.根据人EphA2mRNA基因编码区序列,按照序列设计原则及网上软件,设计并合成两条互补的编码EphA2shRNA的寡核苷酸序列。
2.两条互补的编码EphA2shRNA的寡核苷酸链煺火后与线性载体质粒RNAi-READY-pSIREN-RetroQ连接,构建形成重组质粒pSIREN-RetroQ-EphA2,同时用试剂盒提供的阴性对照siRNA双链寡核苷酸构建对照载体pSIREN-RetroQ-C。
3.以重组质粒pSIREN-RetroQ-EphA2转化大肠杆菌。用碱裂解法少量制备质粒DNA,然后进行酶切鉴定和DNA测序分析。
4.脂质体介导下转染到包装病毒细胞株PA317中,并进行克隆与筛选。
5.以重组质粒pSIREN-RetroQ-EphA2转染MCF-7细胞,并用嘌呤霉素筛选稳定表达的克隆。
6.Westen-blot检测EphA2蛋白的表达。
结果
1.重组逆转录病毒载体pSIREN-RetroQ-EphA2经酶切鉴定结果正确；测序分析证实插入的EphA2 siRNA的方向及序列正确。
2.Western blot分析,与对照空载体及未转染细胞相比,重组逆转录病毒表达载体pSIREN-RetroQ-EphA2转染的MCF-7细胞中EphA2蛋白表达水平明显降低。
结论
1.从EphA2 mRNA序列中筛选出有效的siRNA序列,并设计了互补双链寡核苷酸发夹结构,成功构建了EphA2 siRNA的逆转录病毒表达载体,并将之转染到MCF-7细胞。
2.通过逆转录病毒载体介导的RNAi,能够抑制MCF-7细胞中EphA2勺蛋白表达,为观察转染siRNA的逆转录病毒表达载体的乳腺癌细胞恶性生物学行为变化奠定了实验基础,为以EphA2为靶向的乳腺癌的基因治疗提供了新的思路和手段。
Breast carcinoma is one of the most common malignant tumors and has become the number one killer among women cancers.which greatly affects human health and life.It is estimated that 1.2 million of female will be diagnosed of breast carcinoma, with 0.5 million death annually in the world. The incidence and mortality of breast cancer are increasing, with an annual rate of approximately 0.2～0.3%.And more and more young women were affected by this disease.Classical clinical pathological factors such as age, stage of disease, volume, lymphatic spread and microvascular intensity have been confirmed to have a close relationship with prognosis.And disease recurrence and metastasis in early stage are key factors affecting the outcome of initial therapy. Identification of sensitive biomarkers for recurrence and metastatic potential and their regulatory genes in breast cancer cells, therefore, is important for both early clinical detection and effective prophylactic therapy.
As the rapid development of immunological and molecular biological technologies in the past few decades, the clinical and pathological significance of many biomarkers such as c-er-B-2,ER, PR, uPA,VEGF, BRCA, muc-1,CD44, maspin, mdrl,etc,have been studied in breast cancer at cellular and molecular levels. Among all these biomarkers, c-erbB-2(HER-2), ER and PR are widely studied and considered to be of important prognostic values.But using c-erbB-2,ER and PR as diagnosis and prognostic markers are still not satisfactory enough. However, up to now, it is still mandatory to find better survival prognostic markers to better understand the biological behavior of breast carcinoma, supply with better clinical management of breast carcinoma.
Recently, more and more investigators have focused on the roles of Eph family in tumours, which extends a new wide area for deep researches of the potential mechanisms of signal transduction in carcinomas.The Eph (erythropoitin producing hepatocellular carcinoma) receptor family is the largest subfamily of receptor protein tyrosine kinases (RTK),consisting of at least sixteen distinct receptors and nine membrane-bound ligands, known as Ephrins(Eph family receptor interacting proteins).Signal conduction mediated by Eph receptors and their ligands Ephrins is important to cell-cell repulsion and adhesion, which is also the base of the formation, patterning and plasticity of tissue. Functional studies have demonstrated that Eph receptors and ephrins play important roles in many developmental processes including formation of neuronal network, patterning of neural tube and paraxial mesoderm, guidance of cell migration and axon pathfinding, and vascular formation. Recent studies have also suggested that Eph receptors and ephrins may be involved in carcinogenesis.Recently, gene targeting experiments and angiogenesis assay in vitro and vivo indicates that the genes for Eph receptors and ephrins have been recognized to be differentially expressed in various human tumors.The aberrant expression is associatied with the altered tumor behavior, such as increased invasiveness, increased metas;tatic potential,prominent vascularization, and thus a poor prognosis.
EphA2 was a number of Eph subfamily sifted from human horny cDNA library by Linsberg in 1990,it was the first gene discovered with tyrosine kinase active in Eph subfamily. EphA2 is a transmembrane receptor tyrosine kinase that is found at low levels on adult epithelial cells and enriched within sites of cell-cell adhesion and has been implicated in neuronal development, as well as regulating cell migration and adhesion.Compared with other members of Eph family, EphA2 has been shown frequently overexpressed in multiple types of carcinoma tissues and cell lines, such as, mammary cancer, malignant melanoma, prostatic carcinoma and esphageal carcinomas, et al.Many investigations suggest that EphA2 has a important role in the regulation of angiopoiesis, cell proliferation, invasion and metastasis of carcinoma. It has been proved that the high level of EphA2 can induce the maglignant transformation of mammary epithelial cells in vitro and vivo and it can also promote the invasion and metastasis of the carcinoma. Interestingly, the test of targeting inhibition of EphA2 protein in vitro have shown that using anti-EphA2 antibody can down-regulates expression of EphA2 and inhibits tumour cell growth, and thus lower the malignant degree of the tumor.
EphrinAl (B16) is the major and also the first ligand discovered for EphA2 and was originally cloned from human umbilical vein endothelial cells as a factor induced by tumor necrosis factor-a(TNF-α).Ligand binding contributes to EphA2 autophosphorylation. However, little is known about its expression pattern in tumours. EphA2 and EphrinAl co-overexpressed in many malignant carcinomas and it is reported that the expression of EphA2 correlates with EphrinAl expression. Some study have reported that activation of endogenous EphA2 kinase stimulated by EphrinAl ligand can suppresse integrin-regulated cell adhesion, decreasing cell-cell attachment, endows tumour cells with more aggressive ability. Meanwhile,they found that cell adhesion mediated by integrin is mainly determined by density of EphrinAl ligand. All of these studies indicated that EphA2 and EphrinAl may be involved in the process of tumorigenesis and development of tumours.Recent research reported that the overexpression of EphA2 is related to histological grade, lymph node metastases, and poor prognosis, Suggesting its potential prognostic value in tumor treatment. But recent investigations have indicated that the relationships between overexpression of EphA2 and all sorts of clinicopathological features were different in different typies of tumours.For example, high levels of EphA2 protein in lung cancer predict a high risk of brain metastasis of patients, however, high levels of EphA2 protein did not relate to metastasis in prostatic carcinomas.However, few reports concerned with the expressions of EphA2 and EphrinAl in breast carcinoma in literature in our country is available.
Known and suspected causes for oncogenesis and development of breast cancer include high estrogen level and low progestin level.Metabolic disorder of steroid hormone promotes the development of some kinds of breast cancer, but the involved regulation mechanism remains as yet unclear. Evidence is accumulating that the changes of EphA2 in breast carcinoma interact with ER. But there is little literature about the combined detection of the protein of EphA2,EphrinAl,ERα, ERβand PR in breast carcinoma.
Neo-adjuvant chemotherapy is a method of therapy for non-metastatic cancer that involves systemic treatment with the application of cytotoxic drugs before the local treatment.Currently, the new adjuvant chemotherapy has become the standard treatment for locally advanced breast cancer. The results of the international multi-center randomized clinical trial on neo-adjuvant chemotherapy for breast cancer showed that:Neo-adjuvant chemotherapy is as useful as adjuvant chemotherapy for malignant tumors.The patients with breast cancer that are sensitive to neo-adjuvant chemotherapy have a better prognosis than those who are not sensitive to it. And, good results with the application of the therapy in patients with locally advanced breast carcinoma have already been obtained.With the neo-adjuvant chemotherapy treatment, many pathological and biological indicators of breast carcinoma have changed, and the expression of many genes were altered too.To find out the molecular markers which can predict the efficacy of neo-adjuvant chemotherapy and help to select effective endocrine therapeutic regimen, chemotherapy plan, and to determine the prognosis of breast carcinoma has become a hot spot in the current study. While recent researches on expression of EphA2 and ER, PR in breast carcinoma before and after neoadjuvant chemotherapy is few overseas, and less in China.
Using immunohistochemistry technology to detect the protein expression of EphA2, EphrinAl,ERα, ERβand PR in breast carcinomas, and then studying their relation to clinicopathological factors;The expressions of ER、PR and EphA2 were detected by immunohistochemical method in breast cancer tissues before and after Neo-adjuvant Chemotherapy. We systematically analyzed the expression patterns of EphA2 and EphrinAl in a series of breast carcinomas by RT-PCR technology, explored the relationship between mRNA expressions of EphA2 and EphrinAl and clinicopathological features;In addition, suppression of the expression of EphA2 in MCF-7 cells by using retrovirus-mediated RNAi method was also conducted, In order to find the effective indicators that can help to select effective strategy for treatment and predict prognosis, and to explore the mechanism and prognostic value of EphA2 in breast carcinomas, and to provide new ideas and theoretical basis for gene therapy for breast cancer.This study is divided into four parts as listed below:
Part One:Protein Expressions of EphA2, EphrinA1, ERα, ERβ, and PR in Breast Carcinomas and Their Significance
Objectives:
To study the protein expressions of EphA2,EphrinAl, ERα, ERβ,and PR in breast carcinomas and to explore their significance.
Methods
1.Using Immunohistochemistry Sp method to detect the protein expressions of EphA2, EphrinAl, ERα, ERβ, and PR in tumors from 130 breast carcinomas. Then studying their relationship to clinicopathological factors.
2.The SPSS version 13.0 Statistical package was used for the statistical analysis. We also usedχ2-test(chi-square), t-test(studentt), ANVOA method, and other correlated tests.values of<0.05 were considered statistically significant.
Results
1.The distribution of EphA2,EphrinAl,ERa, ERP and PR was identical, EphA2 and EphrinAl are mainly located in the cytoplasm of tumor cells and endothelial cells and less in the cytoplasm of inflammatory cells. Positive expressions of ERα, ERβand PR were observed in the nucleus of the tumor cells.
2.Among the 130 breast carcinoma cases,94 cases for EphA2 protein were positive,total positive rate was 72.31%;For EphrinAl protein expression,77 cases were positive, total positive rate was 59.23%;For ERαprotein expression,82 cases were positive, total positive rate was 63.08%;For ERβprotein expression,69 cases were positive, total positive rate was 53.08%;67 cases for PR protein expression were positive,total positive rate was 53.08%.
3.The positive expression of EphA2 protein were not associated with age, tumor size and pathological type(P>0.05);But with clinical stage,lymph node metastasis and histological grade(P<0.05).The expression of EphA2 protein of the group with late clinical stage,lymph node metastasis, and high histological grade was respectively higher than the group with early clinical stage, negative lymph node metastasis and low histological grade.
4.The positive expression of EphrinAl protein were not related to age, tumor size, pathological type and lymph node metastasis(P>0.05),but significantly correlated with clinical stage and histological grade(P<0.05).The expression of EphrinA1 protein of the group with late clinical stage, high histological grade was respectively higher than the group with early clinical stage and low histological grade.
5.The protein expressions of ERa and PR were not associated with age,tumor size,pathological type, clinical stage, lymph node metastasis and histological grade(P >0.05).
6.The positive expression of ERβprotein were not associated with age, tumor size, pathological type, clinical stage,and lymph node metastasis(P>0.05), but with histological grade(P<0.05).The expression of ERβprotein of the group with high histological grade was higher than the group with low histological grade.
7.The positive staining of EphA2 and EphrinAl protein co-located in roughly the same tumor areas and vascular endothelial cells.Their distribution are fundamentally unanimous and their expression were positively correlated (P<0.05).
8.Their were no significant correlations between the positive expression of EphA2 protein and the positive expression of PR protein(P>0.05).The exprssion of EphA2 protein was significantly correlated with the expression of ER in breast carcinoma(P<0.05),the positive rate of EphA2 protein expression decreased when the positive rate of ERa protein increased.The positive rate of EphA2 protein in ERa protein-negative group was significantly higher than ERa protein-positive group.The exprssion of EphA2 protein was significantly correlated with the expression of ERβin breast carcinoma(P<0.05),the positive rate of EphA2 protein expression increased when the positive rate of ERβprotein increased.
Conclusions
1.Through the study, we found that EphA2,EphrinA1,ERα, ERβand PR expressed in breast cancer, it is indicated that they may be involved in carcinogenesis, development procedures of breast carcinoma.
2.The positive expression of EphA2 protein were associated with clinical stage, lymph node metastasis and histological grade.The protein expression of EphrinA1 were related to clinical stage and histological grade.The positive expression of ERβprotein were associated with histological grade.These indicated that they may have important potential prognosis value and may be used as new markers to the prognosis evaluation of breast carcinoma. The overexpression of EphA2 is associated with lymph node metastases, indicating the possibility that it may be involved in metastasis of breast carcinoma.
3.Consistent pattern of protein expressions of EphA2 and EphrinAl in breast carcinoma reveals EphrinAl is one of the major ligands which interacted with EphA2 receptor, and EphA2 and EphrinAl could coordinate with each other in signal conduction in breast carcinoma. EphA2 and EphrinAl colocalized in tumour cells and vascular endothelial cells in breast carcinomas, suggesting that EphA2 and EphrinAl may may be invovled in tumour angiogenesis, and therefore could be attractive new targets for breast cancer therapy.
4.Protein expression of ERP and ERa in breast carcinomas suggesting that breast cancer is a kind of hormone-dependent tumor, and we can give directions to clinical endocrine treatment in accordance with the receptor content.
5.There were significantly correlations between EphA2 and EphrinA1,and hinted the expression of EphrinAl was induced by EphA2,EphrinA1 acted on its receptor EphA2 in an autocrine and/or paracrine manner to promote the growth of tumor cells and proliferation of endothelial cells.
6.The protein exprssion of EphA2 were significantly negatively correlated with the expression of ERa protein, and positively correlated with ERβprotein in breast carcinoma(all P<0.05),It old us that EphA2 may be negative regulated by ERa, and may be positively regulated by ERβ.
7. Detecting of the expression of EphA2 and EphrinA1 may be valuable for evaluating the prognosis of breast carcinoma.
Part two:Expressions of ER、PR and EphA2 in Breast Carcinomas with Neoadjuvant Chemotherapy and Their Significance
Objectives
To study the influence of neo-adjuvant chemotherapy on expression of ER、PR and EphA2 in breast cancer and their clinical sigficance.
Methods
The expressions of ER、PR and EphA2 were detected by SP immunohistochemical method in breast cancer tissues of 52 patients before and after Neoadjuvant Chemotherapy.
Results
The expressions of ER、PR and EphA2 were significantly related to treatment response(P<0.05).The positive rate of EphA2 in breast cancer tissues after neoadjuvant chemotherapy was significantly lower than that before the treatment(P<0.05),but The difference of ER、PR before and after Neoadjuvant chemotherapy were insignificant(P>0.05).
Conclusions
Neoadjuvant chemotherapy could inhibit tumor proliferation and induce tumor cell apoptosis,and significantly decrease the expression of EphA2,but it could not reduce the expression of ER and PR significantly.
Part Three:The expressions of EphA2 mRNA and EphrinAl mRNA in breast carcinomas and their significance
Objectives:
To study the expressions of EphA2 mRNA and EphrinAl mRNA in breast carcinoma and their significance
Methods
1.Frozen tissues from 40 cases in which the tumours showed different grades of EphA2 and EphrinAl protein expressions were used for extraction of mRNA.
2.The levels of EphA2 and EphrinA 1 mRNA were detected by RT-PCR and compared if the levels of mRNA correspond to their levels of protein and clinicopathological features.
3.Statistical analysis:All the data were analyzed by the SPSS version 13.0 statistical package.We have used t-test(student t),One-way ANVOA and Nonparametric test(Kruskal-Wallis test method).P values of<0.05 were considered statistically significant.
Results:
1.The positive expressions of EphA2 mRNA were associated with histological grade,lymph node metastasis and clinical stage(P<0.05),But not with age,tumor size and pathological type(P>0.05).
2.The positive expressions of EphrinAl mRNA were associated with histological grade, lymph node metastasis and clinical stage(P<0.05),But not with age,tumor size and pathological type(P>0.05).
3.All the 40 breast carcinoma specimens with different grade EphA2 protein expression have expressed EphA2 mRNA.The mRNA expression levels in those 10 cases with negative protein expression (0.34±0.14) were obviously lower than those 30 cases with 1～3 grade protein expression(protein gradel:1.33±0.25,grade 2: 1.55±0.11,grade 3:1.71±0.19,each compare with grade 0:all P<0.05).Among the 22 cases with protein expression form grade 2 to 3,There were no significant difference in their mRNA expression levels(P>005).Indicating that EphA2mRNA expression did not fully correspond to its protein expression in breast carcinoma.
4. All the 40 breast carcinoma specimens with different grade EphrinAl protein expression have expressed EphrinAl mRNA.The mRNA expression levels in those 7 cases with negative protein expression(0.41±0.11)were obviously lower than those 33 cases with 1～3 grade protein expression(protein gradel:1.37±0.38, grade 2: 1.59±0.28,grade 3:1.67±0.19, each compare with grade 0:all P<0.05).Among the 21 cases with protein expression form grade 1 to 2,There were no significant difference in their mRNA expression levels(P>005).Among the 24 cases with protein expression form grade 2 to 3,There were no significant difference in their mRNA expression levels(P>005).Indicating that EphrinA1 mRNA expression did not fully correspond to its protein expression in breast carcinoma.
Conclusions
1.The positive expressions of EphA2 mRNA and EphrinAl mRNA in breast carcinoma were associated with histological grade, lymph node metastasis and clinical stage(P<0.05).This indicated that the positive expressions of EphA2 mRNA and EphrinAl mRNA were related to the malignant biological properties of breast carcinoma. And indicating that they may become new markers to the evaluation of the prognosis of breast carcinoma.
2.To some extent, the positive expression of EphA2mRNA and EphrinA1 mRNA in breast carcinoma were associated with the expression of EphA2 protein and EphrinAl protein respectively, indicating that they were both regulated at transcriptional level.
3.The positive expression of EphA2mRNA and EphrinAlmRNA in breast carcinoma were partly different from the expression of EphA2 protein and EphrinAl protein respectively, indicating that there may be some post-transcriptional and translational regulation mechanisms, some accumulation obstacles and some degradation pathway disorders.
Part four:Study of targeted silencing of EphA2 in breast cancer cell line MCF-7 by RNA interference
Objectives
To investigate the effects of retrovirus-mediated siRNA targeting EphA2 on the breast cancer cell line MCF-7, in order to provide the possibility of theoretical basis of gene therapy in vitro for breast cancer.
Methods
1.Based on the human EphA2mRNA gene sequencing and according to the design principle, two complementary oligodeoxyribonucleotides encoding EphA2 short hairpin RNA were designed and synthesized with the software online.
2.Then these two complementary oligodeoxyribonucleotides were annealed and ligated into a linear vector RNAi-READY-pSIREN-RetroQ to construct the recombinant plasmid pSIREN-RetroQ-EphA2.The control vector (pSIREN-RetroQ-C) was constructed at the same time which contain a free correlation sequence.
3.The ligation mixtures were transformed into competent E.coli DH5a. And the recombinant plasmids pSIREN-RetroQ-EphA2 were extracted from small-scale bacterial cultures by Alkaline Lysis, Subsequently the plasmids were digested and then identified by DNA sequence analysis.
4.The vectors based RNAi were transfected into packing cell line PA317,which was selected by puromycin later.
5.MCF-7 cells were transfected by recombinant plasmids pSIREN-RetroQ-EphA2 and stable transfectants were isolated by puromycin.
6.The EphA2 protein in transfected MCF-7 cells was detected by Western blots.
Results
1.The pSIREN-RetroQ-EphA2 vector was confirmed by restriction endonuclease digestion and It was verified by DNA sequencing that the insert sequence was successfully cloned into the vector.
2.Compared with cells transfected with empty retroviru vector and untransfected cells, the level of EphA2 protein expression was greatly decreased in MCF-7 cells transfected by the recombinant retroviru vector by Western blots techniques.
Conclusions
1.We selected effective EphA2 siRNA sequence from EphA2 mRNA sequence and designed and synthesized the hairpin structure of double complementary chains of oligonucleotides,The recombinant retro viral vectors were constructed successfully and retroviru has been successfully transfected into MCF-7 cells line and it works well.
2.EphA2 protein expression in MCF-7 cell line is successfully suppressed by using the recombinant retroviru-mediated RNAi technique, indicating that this may contributes to the study of the changes of malignant biological activity of breast cancer cell lines by transfection of siRNA retrviral vectors and provide a new solution for EphA2-targeting therapeutic intervention of breast carcinoma.
引文
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