【作者】 何文山；

【导师】 王春友； 黄韬；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 目的乳腺癌是常见的女性恶性肿瘤,在欧洲及北美等发达地区,乳腺癌的发病率长期位列女性恶性肿瘤的第一位。随着经济的发展,我国大城市和沿海发达地区的发病率也开始呈现迅速上升的趋势,其发病率也已成为女性所有恶性肿瘤中的第一或第二位,严重威胁女性健康。目前在临床上治疗乳腺癌的手段主要有手术治疗、化疗和内分泌治疗,分子靶向治疗刚刚崭露头角,但是相较于传统的治疗手段,它已经显示出巨大的潜在价值。所以,寻找能够对乳腺癌进行早期诊断,或能准确判断其预后,以及能为分子治疗提供新靶点的基因,对于乳腺癌基因治疗的发展和推广就显得尤为重要。目前在分子肿瘤学领域的热点之一就是对肿瘤细胞周期的研究,细胞周期蛋白家族(Cyclins)自然而然就进入了广大研究者的视野,而其中的细胞周期蛋白E(Cyclin E)作为一个已经确定的癌基因,作为细胞周期中G1/S期的关键调节蛋白,其研究价值正日渐浮出水面。近年来,很多国内外乳腺癌疾病的研究者都注意到了Cyclin E,尤其是低分子量剪切型Cyclin E(LMW Cyclin E)同乳腺癌预后之间的密切关系。本课题正是从Cyclin E在乳腺癌组织中的表达水平入手,总结Cyclin E的表达同乳腺癌患者预后之间的关系。接着以RNAi为干预手段,设计可以抑制Cyclin E表达水平的实验工具,在体内及体外水平分别抑制乳腺癌中Cyclin E的表达,进而分析Cyclin E对肿瘤生物学行为的影响并试图挖掘Cyclin E作为一个乳腺癌基因治疗新靶点的潜在价值。方法收集华中科技大学同济医学院附属协和医院病理科2000-2003年部分乳腺病检蜡块,结合其相应的临床资料,按国际抗癌学会(UICC)及美国癌症联合会(AJCC)建议的TNM分期法(第六版)对其进行临床分期。其中乳腺良性肿瘤18例,恶性肿瘤(乳腺癌)80例,平均年龄50岁,所有患者于手术前未接受任何抗癌治疗。用免疫组织化学法检测上述研究对象中Cyclin E蛋白的表达,以及C-erbB-2(HER-2/neu)、nm23-H1、actin(肌动蛋白)的表达。根据Cyclin E cDNA序列设计引物。再依照载体法合成siRNA的原则选择2段靶序列,设计并合成Cyclin E基因的siRNA片段,退火后于pGENSIL-1载体连接,构建Cyclin E基因的siRNA真核表达载体。再用该载体瞬时转染人乳腺癌细胞株MCF-7,检测转染前后对照组及转染组中Cyclin E mRNA的表达与蛋白水平的表达,CCK-8法检测siRNA对细胞的抑制效应,流式细胞术检测细胞周期。分别用对照组及转染组MCF-7细胞建立裸鼠移植瘤模型,绘制肿瘤生长曲线。取一部分荷瘤裸鼠模型以siRNA进行肿瘤局部注射,模拟基因治疗,并绘制肿瘤生长曲线。结果Cyclin E在良、恶性肿瘤组织中过表达的差异性有极显著的统计学意义(χ2=13.29,P<0.01),即在良性乳腺肿瘤中Cyclin E的过表达率很低,而在乳腺癌组织中Cyclin E的过表达率较高;而在乳腺癌的不同组织类型中Cyclin E过表达的差异性没有统计学意义(χ2=0.1099,P>0.05)。在临床分期较早的乳腺癌组织中Cyclin E的过表达率较低,而在临床分期较晚的乳腺癌组织中Cyclin E的过表达率较高,且随着临床分期的进展,Cyclin E的过表达率也相应地升高。ER阳性的乳腺癌组织中Cyclin E的过表达率较ER阴性者为低,其Cyclin E过表达率的差异具有显著的统计学意义(χ2=4.1112,P<0.05)。在PR阳性或阴性的乳腺癌组织中,Cyclin E阳性表达率的差异性没有显著的统计学意义(χ2=1.3267,P>0.05)。在C-erbB-2(HER-2/neu)呈阳性表达乳腺癌组织中,Cyclin E的阳性表达率较高;而在C-erbB-2(HER-2/neu)呈阴性表达的乳腺癌组织中,Cyclin E的阳性表达率较低,这一阳性表达率的差异性有着显著的统计学意义(χ2=4.0635,P<0.05)。在nm23-H1表达呈阳性和阴性的乳腺癌组织中,Cyclin E的阳性表达率的差异性没有显著的统计学意义(χ2=1.6737,P>0.05)。actin呈阴性表达或是呈不连续分布即恶性度高的乳腺癌组织同actin呈阳性表达且分布连续的恶性度低的乳腺癌组织相比,其Cyclin E的阳性表达率的差异有着显著的统计学意义(χ2=4.2092,P<0.05)。成功地设计并合成了序列ctrl,CCNE-1和CCNE-2,其测序结果与我们设计合成的靶向Cyclin E的反义链完全一致,并将上述目的基因正确地插入pGENESIL-1载体,成功构建重组质粒。转染24 h后的转染效率约50%。转染48h后实验组细胞内Cyclin E的mRNA RT-PCR产量明显低于空白对照及阴性对照组。siRNA作用于细胞后,Western Blot检测显示Cyclin E蛋白水平的表达有显著降低。经化疗药物处理后,转染后细胞的抑制率较未转染细胞有明显上升,且这一差异有统计学意义。转染siRNA后,乳腺癌细胞被大量阻滞于G1期。动物实验方面,对照组裸鼠移植瘤生长速度较转染组快,瘤体体积也较大。空白对照组和生理盐水对照组中裸鼠移植瘤的生长较siRNA局部注射治疗组迅速,肿瘤体积也较大。结论在一定程度上,Cyclin E的过表达可以反映乳腺的良、恶性病变的差异,Cyclin E的表达与乳腺癌临床分期密切相关,即由Ⅰ期至Ⅳ期阳性表达率逐渐升高,或可将其作为一个判断乳腺肿瘤预后的分期性指标。抑制乳腺癌细胞中Cyclin E的表达可以显著抑制肿瘤细胞的生长,提高肿瘤对化疗的敏感性,抑制肿瘤细胞的成瘤能力,Cyclin E siRNA对肿瘤生长亦具有抑制作用。Cyclin E具有成为乳腺癌基因治疗新靶点的潜在价值。更多还原

【Abstract】 Objective Breast cancer is a common women malicious disease. In Europe and North America, the incidence of breast cancer has been the first place of all the women malicious diseases for many years. As the development of economy, the incidence of breast cancer began to raise quickly in some developed littoral area of China, it has already been the first or second place of all the cancers of women. Breast cancer seriously threatened women health. At present, the major clinical treatments to breast cancer are surgery, chemical therapy and endocrine therapy. Gene therapy has emerged recently, it showed tremendous potential ability to treat breast cancer. So, it is very important to find some new genes which can early diagnose breast cancer, or predict the prognosis of breast cancer, or provide a target for gene therapy. Lately, the hottest spot in molecular oncology is the research of cell cycle, and Cyclins are the most popular gene family in this field. As a oncogene and a gatekeeper gene of G1/s phase in cell cycle, Cyclin E, especially LMW Cyclin E has been noticed for the strong relationship to breast caner prognosis by many researchers. This study measured the expression of Cyclin E in breast cancer, conclude the relationship between Cyclin E expression and breast cancer prognosis. Then we designed siRNA sequences according to RNAi technology to inhibit the expression of Cyclin E in vivo and in vitro, and analysed the influence of Cyclin E to breast cancer’s biology behavior, and finally discovered the potential value of Cyclin E as a new taget to gene therapy.Methods Tumor tissues were obtained from the pathology department of Huazhong University of Science and Technology Tongji Medical College Union Hospital. Each patient had received a diagnosis of breast cancer according to the TNM method which is recommended by UICC and AJCC between 2000 and 2003. Among them there are 18 cases of benign tumor and 80 cases of breast cancer. All patients have not accepted any treatment before surgery. We examined the expression of Cyclin E, HER-2/neu, nm23-H1 and actin by immunohistochemical methods. We designed and synthesized two pairs of siRNA according to the Cyclin E cDNA sequence in Genebank, then inserted them into pGENESIL-1 plasmids respectively. The recombinants (named pGENESIL/Cyclin E-1 and pGENESIL /Cyclin E-1) were sequenced and identified. We transfected the vector into MCF-7 cell line, RT-PCR and Western blot were performanced to examine the expression of Cyclin E in mRNA and protein level respectively. Cell cycle was measured by FCM. And after being treated with chemotherapy drugs, the MCF-7 growth and proliferation were examined by CCK-8 assay. The nude mice bearing human breast cancer were prepared with MCF-7 and transfected MCF-7 and Cyclin E siRNA was injected to nude mice bearing human breast cancer. The growth of tumor was observed.Results The over expression rate of Cyclin E in malignant tissues is obviously higher than that in benign tumor tissues, P<0.01. The over expression of Cyclin E in later stage of disease is higher than that in early stage of disease, P<0.05.The expression of Cyclin E in ER positive tissues is lower than that in ER negative tissues, P<0.05. The expression of Cyclin E in PR positive tissues and in PR negative tissues has no significant different, P>0.05. The expression of Cyclin E in HER-2/neu positive tissues is higher than that in HER-2/neu negative tissues, P<0.05. And the expression of Cyclin E in ER, PR and HER-2/neu all positive tissues is much higher, P<0.01. The expression of Cyclin E in nm23-H1 positive tissues and in nm23-H1 negative tissues has no significant different, P>0.05. The expression of Cyclin E in actin positive and continuous distribution tissues is lower than that in actin negative or uncontinuous distribution tissues, P<0.05. Cyclin E siRNA eukaryotic expression vectors were succesfully constructed. Sequence analysis of inserted fragments revealed the same sequences as synthesized siRNA oligonucleotides. RT-PCR and Western blot showed the expression of Cyclin E was significantly lower after MCF-7 was transfected. After being treated by chemical drugs, the growth and proliferation of transfected MCF-7 was lower then that of non-tranfected cells, and there was a significant difference. And its cell cycle was arrested at G1 phase. The tumor forming ability of transfected MCF-7 was lower than that of non-transfected MCF-7. Cyclin E siRNA could inhibit the growth of tumor beared by nude mice.Conclusions To some extend, the over expression of Cyclin E can reflect the difference between benign and malicious breast diseases. The level of Cyclin E expression has a strong relationship with the clinical phases of breast cancer. The expression of Cyclin E can be a division index of breast cancer prognosis. Inhibition of Cyclin E can obviously depress cancer cells’ proliferation, raise their sensibility to chemical therapy and restrain their tumor forming ability. Cyclin E siRNA can be used to inhibit the growth of breast cancer cells. Cyclin E has the potential to be a new target for gene therapy of breast cancer.更多还原