【作者】 吕辉；

【导师】 文继舫； 吴晓英；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 研究背景及目的：乳腺癌是世界范围内妇女最常见的恶性肿瘤之一,严重威胁着女性的身心健康和生命。乳腺癌的发生为多基因参与和多阶段协同作用的结果,其中细胞内癌基因的活化和/或抑癌基因的失活是导致乳腺癌发生的重要原因。研究新的抑癌基因对乳腺癌产生的作用及引起的生物学特性的改变,对于阐明乳腺癌的发病机制、指导临床治疗具有重要意义。研究发现,在10-30%的乳腺癌中检测到肿瘤细胞出现神经内分泌分化现象,而这一现象出现的原因及意义仍不十分明确。抑制元件-1沉默转录因子REST在正常上皮组织中广泛表达,具有抑制细胞中神经内分泌相关基因表达的作用,其功能缺失与前列腺癌细胞出现神经内分泌分化有关；最近有研究报道REST具有抑制肿瘤发生的作用,REST基因缺失或突变与小细胞肺癌、前列腺癌、肠癌等许多肿瘤的发生密切相关,将正常乳腺上皮细胞中REST功能抑制后可引起细胞出现锚定非依赖性生长的恶性转变。那么,REST功能异常是否与乳腺癌的发生有关,是否参与了乳腺癌细胞神经内分泌分化的过程,目前国内外未见相关报道。本研究通过分析REST在乳腺癌标本中的表达及意义、揭示REST在乳腺癌细胞中的作用,以期进一步完善乳腺癌的发病机制,为乳腺癌的治疗提供新的靶标及为乳腺癌的预后判断提供新的科学依据。方法：收集乳腺正常组织、良性病变及乳腺癌标本,通过免疫组织化学的方法检测各组织中REST、Syn、ER、PR、Her-2蛋白的表达情况。采用χ2检验,Spearman’s等级相关分析等统计方法,分析REST在乳腺癌的发生、神经内分泌分化及转移中的可能作用。进一步研究REST在乳腺癌细胞系中的作用,首先,采用RNA干扰技术稳定干扰内源性高表达REST细胞中REST的表达,通过RT-PCR、Western Blot等方法鉴定稳定干扰REST的细胞系；生长曲线、平板克隆及软琼脂集落克隆形成率检测细胞的增殖；Hochest凋亡染色、流式细胞术检测细胞凋亡；单细胞凝胶电泳、MTT药敏试验检测细胞对化疗药物的敏感性。其次,通过外源性REST转染重建REST低表达细胞中REST的表达,流式细胞术检测细胞凋亡改变；透射电子显微镜技术检测细胞损伤情况；侵袭小室检测细胞的迁移与侵袭能力改变。Western Blot检测与增殖、凋亡及侵袭相关基因的表达情况。结果：免疫组织化学结果显示,乳腺癌组织中REST的阳性表达率为69.1%,较非癌性病变的乳腺组织明显下降,差异具有统计学意义(p<0.05)。同时,在乳腺癌标本中比较分析腋窝淋巴结转移组与未转移组,发现前者REST的表达阳性率也显著低于后者；本研究检测的68例乳腺癌组织中有22.1%的乳腺癌组织出现神经内分泌分化现象,而且乳腺癌组织神经内分泌分化现象的出现与REST蛋白的表达呈负相关(p<0.01,R=-0.412)；REST蛋白的表达与ER、PR及Her-2的表达无明显关联(p>0.05)；稳定沉默内源性高表达REST的乳腺癌细胞MCF-7中REST基因表达后,细胞生长加快,细胞周期G1/S期进程加速,细胞克隆形成能力及停泊非依赖性生长能力增强,与增殖有关的蛋白PI3K表达上调,Akt蛋白磷酸化水平升高；稳定沉默MCF-7细胞中REST的表达后,细胞的凋亡抗性增加,细胞内与凋亡相关的蛋白Caspase-7活性降低、c-Jun蛋白的磷酸化水平下降；肿瘤细胞对化疗药物敏感性下降,抗凋亡基因Bcl-2表达上调；重建REST低表达细胞MDA-MB-231细胞中REST的表达后,乳腺癌细胞的凋亡率上升；化疗药物作用后,细胞的损伤程度加重；增强MDA-MB-231细胞内REST的表达后,乳腺癌细胞的迁移、侵袭能力受到抑制,而且细胞内金属基质蛋白酶MMP-9的表达下调。1、首次揭示REST:在乳腺癌中的表达下降,证明REST表达下降与乳腺癌的转移及乳腺癌细胞神经内分泌分化的发生有关；且NED可能促进肿瘤的转移；2、REST功能缺失后通过活化PI3K/Akt信号通路,促进细胞生长增殖；通过抑制Caspase-7和c-Jun的活化增加细胞的凋亡抗性；说明在乳腺癌细胞中REST蛋白具有抑制细胞增殖和启动细胞凋亡等抑瘤功能,3、REST沉默后可上调抗凋亡基因Bcl-2的表达介导乳腺癌细胞耐药,提示REST可能与肿瘤耐药有关。4、首次证实REST蛋白通过下调金属基质蛋白酶MMP-9的表达进而抑制乳腺癌细胞侵袭转移。更多还原

【Abstract】 Background and Objective:Breast cancer is the most common cancer of women and the second leading cause of female cancer mortality in the world. The mortality rate of breast cancer could be lowered by identification of novel molecular markers of early breast cancer and understanding the molecular mechanisms of the early events of breast cancer. It has been reported that there are 10-30% breast cancer show the character of neuroendocrine differentiation(NED). But the mechanism is still unclear. RE1 silencing transcription factor (REST) is generally expressed in all of epithelial cell, who in charge of the expression of neuronal gene. Dysfunction of REST took part in the NED of prostate cancer. And recently, the tumor suppression role of REST is identified. REST expression was recently shown to be absent in many other cancers, such as small cell lung cancer (SCLC), colorectal cancer and prostate cancer. Blockade of REST function in the epithelial cells is found to cause a transformation phenotype, such as anchorage-independent growth. Neither REST take part in the development of breast cancer nor REST may have relationship with the NED of breast cancer has been reported.The aim of this study is to investigate the expression of REST in breast cancer and analysis the possible role of REST in breast cancer. The results could contribute to elucidate the mechanism of tumorigenesis. It could provide a new target for the treatment of breast cancer. And also provide a new prognosis factor for breast cancer.Methods:The samples were collected. And the expression of REST, Syn, ER, PR and Her-2 were detected by Immunohistochemistry. The expression of REST and the relationship of them in the breast cancer were analyzed byχ2 test and Spearman’s rank correlation analysis. To further investigate the possible role of REST in breast cancer cell, the endogenous expression of REST was shut-down by RNAi. The cell line in which REST expression was stable silenced was identified by RT-PCR and Western Blot. Growth curve and colony formation were used to detect the cell proliferation. The apoptosis of cell was detected by Hochest 33258 stain and Flow Cytometry. Single cell gel electrophoresis and MTT chemotherapy sensitivity were used to analysis the sensitivity of cell to anticancer drug. Moreover, reconstitution of REST in breast cancer cells by transgene. Flow Cytometry and Transmission electron microscope were used to analysis the apoptosis and damage of cells. The capability of migration and metastasis were detected by Transwell analysis. Western Blot was used to detect the expression of genes that associated with proliferation, apoptosis and metastasis.Results:The positive expression of REST was only 69.1% in breast cancer. The difference was significant between the breast cancer and benign breast samples (p<0.05). Also the positive percent was also declined in breast cancer in which the metastasis was happened. There was 22.1% of breast cancer showed the character of neuroendocrine differentiation(NED). Furthermore, there was negative relationship between the expression of REST and NED (p<0.01, R=-0.412). There was no significant relation among the expression of REST and ER, PR, Her-2(p>0.05).The proliferation of MCF-7 cell, in which the endogenous expression of REST was relatively high, was enhanced when REST was shut down. The expression of PI3K and p-Akt were enhanced. Also the apoptosis of MCF-7cell was reduced when REST was shut down. And concomitance with activity of Caspase-7 and c-Jun were reduced. Loss of REST expression resulted in a reduction chemosensitivity to anticancer drug. And the increasing in Bcl-2 expression was detected.The apoptosis was increased when reconstitution of REST in breast cancer cell MDA-MB-231, in which the expression of REST was relatively low. The metastasis ability of MDA-MB-231 cell was restrained when the expression of REST was up-regulated, in concomitant with the suppression of MMP-9 expression.1. The expression of REST was down-regulated in breast cancer. There was some relationship between the reduced expression of REST and the metastasis of breast cancer. The reduced expressions of REST maybe take part in the development of NED in the breast cancer. And the NED of breast cancer may promote the metastasis of breast cancer.2. The PI3K/Akt pathway was activated when the expression of RSET was shut down. This maybe results in proliferation of cells. And the apoptosis was reduced concomitance with the activation of Caspase-7 and c-Jun was decreased. All of these indicated that the role of REST was suppression of proliferation and induction of apoptosis.3. REST mediated the drug resistance in the breast cancer through increasing the expression of Bcl-2. REST maybe take part in chemotherapy resistance of breast cancer.4. REST attenuated the capability of metastasis of breast cancer cell by suppressing the expression of MMP-9.更多还原