【作者】 王景毅；

【导师】 邵国光；

【作者基本信息】 吉林大学 ， 外科学， 2013， 博士

【摘要】 研究背景和目的乳腺癌是一种常见的、严重威胁人类生命健康的恶性肿瘤。近些年来，随着我国人口老龄化、农村城市化和城镇工业化的进程、人们生活环境污染与破坏、人们生活饮食习惯等生活方式的改变，其发其发病率和死亡率逐年上升，目前在我国城市中居女性恶性肿瘤的首位。转移是影响乳腺癌患者治疗效果和死亡的主要原因。因此探讨与乳腺癌转移相关的因素，阐明乳腺癌转移机制，寻找预防和治疗的有效途径，寻找新的早期诊断和治疗靶点，提高治疗效果是目前乳腺癌研究的重点内容之一。IRF2基因定位于染色体4q34.1-q35.1，表达无组织特异性，在肿瘤调节过程中IRF2具有与IRFl相反的作用，由于IRF1与IRF2的识别位点相同，IRF2可通过竞争结合相同的识别位点而抑制IRFl的转录。过表达IRF2的NIH3T3细胞能使裸鼠致瘤，而过表达IRF2的同时表达IRFl则将消除NIH3T3细胞的致瘤能力。Keller等研究发现IRF2能与Blimpl（B-lymphocyte-inducedmaturation protein1）共同结合于IFN-8基因VRE的PRDI结构域，Blimpl具有促分化抑制增殖作用，而IRF2能抑制Blimpl的作用而引起细胞癌变。在增殖细胞中，IRF2乙酰化后可结合到组蛋白H4的启动子，引起细胞持续增殖。N-ras是细胞生长抑制因子，在骨髓细胞系过表达IRF2可以逆转N-ras诱导的生长抑制。IRF2可通过干扰IRFl与ISRE的结合削弱干扰素的作用，从而促进细胞增殖和癌变。本研究在临床样本中分别从核酸和蛋白水平，探讨IRF2对乳腺癌增殖转移的影响。方法1、乳腺癌组织和细胞中IRF2的表达。首先采用Western blot和RT-PCR技术从蛋白和核酸水平检测IRF2在几种常见的乳腺癌细胞株及临床组织病理标本中的表达情况，初步探讨IRF2与乳腺癌细胞的关系。2、乳腺癌临床组织标本中IRF2的表达检测及其临床意义分析。通过回顾性分析180例乳腺癌组织样本的随访信息和临床病理资料，应用免疫组化技术检测IRF2蛋白的表达情况。分析IRF2蛋白表达与乳腺癌患者生存期的关系，为预后判定提供依据。3、IRF2表达沉默对乳腺癌细胞生物学行为的影响。利用在线数据库和设计软件设计两个个IRF2干扰片段，构建慢病毒载体，将慢病毒载体质粒与辅助质粒共转染包装慢病毒。用含IRF2特异性干扰片段的慢病毒感染MDA-MB-231细胞，以含无关序列载体的病毒为阴性对照。筛选抗性单克隆，RT-PCR和Western blot检测各干扰克隆细胞中IRF2基因干扰效率，以干扰效率最高的克隆为下一步实验的研究对象。利用MTT比色法、平板克隆形成实验及流式细胞仪检测IRF2沉默后对细胞体外增殖的影响；利用划痕实验、体外侵袭小室（boyden小室）进行体外侵袭实验，检测IRF2沉默后对乳腺癌侵袭能力的影响。结果1、乳腺癌组织和细胞中IRF2的表达。RT-PCR方法在核酸水平的检测结果表明相对于正常对照，IRF2基因在几种乳腺癌细胞中的表达水平明显增高；IRF2在20例乳腺癌组织样本中的表达水平也均明显高于其对照配对正常乳腺组织。并且在乳腺癌中IRF2基因表达水平的增高与乳腺癌的病理类型无关，提示在乳腺癌细胞中存在IRF2基因的普遍表达。而Western blot方法对正常乳腺和乳腺癌细胞和组织中IRF2蛋白的表达检测结果与核酸水平的检测结果基本一致。2、乳腺癌组织和细胞中IRF2蛋白的表达及临床意义。通过免疫组化检测结果显示为“浆阳”，即IRF2蛋白主要表达在乳腺癌组织细胞中的胞浆。在180例乳腺癌组织中，IRF2的阳性率为54.44%（98/180）；在100例正常乳腺组织中IRF2阳性率为33.00%（33/100）。IRF2蛋白在不同病理类型的乳腺癌细胞中的表达无显著性差异（P>0.05）；相对于正常对照乳腺组织，IRF2蛋白在乳腺癌组织中表达的阳性率明显增高，差异具有统计学意义（P<0.05）。IRF2蛋白的表达与患者临床分期和有否淋巴转移密切相关（P<0.05）；而IRF2的表达水平和病理类型、肿瘤的大小及患者的性别、年龄无明显相关性（P>0.05），结果表明在乳腺癌组织中IRF2蛋白高水平表达，并与淋巴转移相关。3、IRF2表达沉默对乳腺癌细胞生物学特性的影响。利用RNAi技术建立IRF2基因沉默的稳定乳腺癌细胞株。构建了两个IRF2的慢病毒干扰载体，并包装慢病毒后，含有干扰载体的慢病毒感染MDA-MB-231细胞，杀稻瘟菌素筛选抗性克隆，约14天后，挑取建立抗性单克隆，利用RT-PCR和Western blot技术检测各克隆的IRF2的表达情况，筛选干扰效率最高的克隆为下一步实验的研究对象。MTT比色法观察IRF2基因表达沉默后体外细胞的增殖情况，与MDA-MB-231/mock细胞相比，MDA-MB-231/IRF2细胞的增殖速度明显减慢，并且呈时间依赖关系（P<0.05）。平板克隆形成实验显示MDA-MB-231/IRF2细胞的活力显著下降（P<0.05）。利用流式细胞仪检测各组细胞的细胞周期，结果显示MDA-MB-231/IRF2细胞S期比率增加，表明IRF2表达沉默后细胞增殖减慢。分析结果表明IRF2表达水平减低后，肿瘤细胞体外生长显著减慢。利用划痕实验检测IRF2基因表达沉默后细胞运动能力的改变，实验结果显示，随着细胞培养时间延长，不同时间迁移细胞数有着明显差异（P<0.05）；MDA-MB-231/IRF2细胞迁移数明显低于MDA-MB-231和MDA-MB-231/mock，差异具有统计学意义。结果表明IRF2沉默后MDA-MB-231细胞的运动迁移能力减慢。利用体外侵袭小室实验检测IRF2基因表达沉默后细胞侵袭能力的变化，实验结果发现，MDA-MB-231/mock细胞相比，MDA-MB-231/IRF2细胞的侵袭能力明显减低（P<0.05），说明IRF2表达沉默降低了乳腺癌细胞的体外侵袭能力。结论1、慢病毒干扰载体可以有效地下调IRF2的表达，降低乳腺癌细胞体外侵袭、增殖及转移的能力；IRF2是乳腺癌侵袭和转移过程中的重要调控因子。2、IRF2的表达与乳腺癌患者的预后相关，可能为影响乳腺癌患者生存的独立预后因素。更多还原

【Abstract】 BACKGROUND＆OBJECTIVEBreast cancer is the most common tumor in the world at present. In recentyears, along with the many environmental factor influence, the morbidity rateand mortality rate of breast cancer rase rapidly in the world. The main cause ofdeath in Breast cancer cell patients is the invasion and metastasis of tumors. It iscritical to elucidate molecular mechanisms of metastasis and find the key factorscontrolling tumor metastasis, it will also be helpful to develop the effectivepreventive and therapeutic strategies. So it becomes the present big issue toinvestigate the molecular mechanism of breast cancer cell invasion andmetastasis and to look for novel related molecular marker and target.Interferon regulatory factor-2(IRF2), an important member of thehigh-mobility group protein superfamily, has been implicated in a variety ofbiologically important processes, including transcription, DNA repair, V(D)Jrecombination, differentiation, development, and extracellular signaling. Recentstudies indicated that Interferon regulatory factor-2(IRF2) takes part in type IIFN-regulated gene expression where it forms the transcriptional complexIFN-stimulated gene factor3(ISGF3) with STAT multimers. However, studiesexploring the role of IRF2in breast cancer are scarce. Thus, in this investigationwe studied the effect of lentivirus-mediated RNA interference (RNAi) of IRF2gene in breast cancer cells. METHODS1. Expression analysis of IRF2in Breast cancer cell by real-time PCR andWestern blotTo determine correlation of IRF2with Breast cancer cell, real-time PCRand Western blot analysis were performed to evaluate the expression levels ofIRF2transcripts and protein in five breast cancer cell lines with different tissuesamples.2. Expression of IRF2in Breast cancer and its clinical significanceProtein expression of IRF2in normal breast tissue and breast cancer celland its possible clinical significance in carcinogenesis, progression, invasion andmetastasis were studied by immunohistochemistry.3. Lentivirus-mediated silencing of IRF2gene and the influence on humanbreast cancer cell cellsTwo short hairpin RNAs were designed to silence IRF2expression, andrecombinant lentivirus vectors under the control of the U6promoter with fourshort hairpin RNAs were constructed. T47D cells were co-transfected withrecombinant lentivirus vectors and adjunctive plasmid. Virus supernatants wereharvested and stored in-80℃before used in the next experiments, virus titerwas determined as routine.MDA-MB-231, a kind of human breast cancer cells, were infected withvirus supernatants obtaining IRF2specific RNAi lentiviral vectors,MDA-MB-231infected with virus supernatants obtaining mocked RNAilentiviral vectors were regarded as controls. Cells IRF2stably silenced werescreened by blasticidin. Quantitative RT-PCR and Western blot analysis wereused to examine the effectiveness of RNA interference. Effects of IRF2silence on cell proliferation was assessed by MTT assay, plate colony formation assayand flow cytometry in vitro. Moreover, motility and migration of tumor cellswere determined by scratch-wound healing assay and in vitro Boyden chamberassay.RESULTS1. Expression of IRF2in the samples of cell lines and tissues from humanbreast cancer cells by real time RT-PCR and Western blotThe results showed that the expression levels of IRF2mRNA in the5celllines of human breast cancer cells were higher than that of in cell line originatedfrom breast of human embryo. Compared with paired normal breast tissues, asignificant decreasing in the expression levels of IRF2mRNA was noted in20samples of primary human breast cancer tissue. The under-expression of IRF2gene in human breast cancer tissues was not correlated with the pathologicaltypes of breast cancer. We then examined the protein expression of IRF2proteinin cell lines and tissues of human breast cancer and normal controls. Theexpression level of IRF2protein was consistent with that of mRNA on thewhole.2. Expression of IRF2protein in human breast cancer tissues by immunohistochemistry and its clinical significanceThe positive expression of IRF2was found in54.44%（98/180）of humanbreast cancer tissues. The rate of the positive expression of IRF2protein in100samples of normal breast tissue was33.00%（33/100）. Compared with normaltissues, a significant decreasing in expression of IRF2protein was observed inprimary human breast cancer tissues (P<0.05). No significant associations werefound between IRF2expression and age, gender, tumor size and tumor type of pathology (P>0.05). However, IRF2expression was positively correlated withlymph node metastasis and TNM stage (P<0.05).3. Effects of IRF2silencing on biological behaviors of human breast cancercells.1) Establishment of MDA-MB-231cells stably IRF2silenced by RNAinterferenceWe constructed plasimids that expressed short hairpin RNAs that weretargeted against IRF2by lentiviral vector. Two different sequences wereoriginally selected for targeting IRF2gene. We infected MDA-MB-231cellswith lentival vectors and examined IRF2mRNA and protein expression, andfound IRF2siRNA S1was the most effective at blocking IRF2expression. ThesiRNA S1was selected to be investigated in our next experiments.2) Effects of IRF2silencing on the biological behaviors of humanMDA-MB-231cellsA significantly time-dependent inhibitory proliferation was found inMDA-MB-231/IRF2-cells as compared with MDA-MB-231/mock andMDA-MB-231cells by in vitro MTT assay(P<0.05). In addition, MDA-MB-231/IRF2-cells had a significant impaired ability to form colonies in platesas compared with MDA-MB-231/mock and MDA-MB-231cells(P<0.05).Interestingly, MDA-MB-231/IRF2-cells showed a significantly decreasedamount in S period by flow cytometry. The results indicated that silence of IRF2partially blocked proliferation of MDA-MB-231cells.To evaluate the role of IRF2silencing on movement and migration ofMDA-MB-231cells, we performed in vivo wound healing experiment. Theresults showed that each group had singifcant difference in different time (P<0.05). MDA-MB-231/IRF2-cells had significantly reduced movement andmigration compared with MDA-MB-231/mock and MDA-MB-231cells. Therewas also significant difference in different time among three groups (P<0.05).MDA-MB-231/IRF2-cells had significantly reduced invasiveness ascompared with MDA-MB-231/mock and MDA-MB-231cells as determined byin vitro boyden cave assay (P<0.05), suggesting that IRF2silencing inhibited theinvasive abilities of MDA-MB-231cells.CONCLUSION1. IRF2can promote proliferation and migration of human breast cancercells. IRF2gene significantly correlates to proliferation, invasion and metastasisof human breast cancer cells.2. IRF2expression might be an independent prognostic indicator for thesurvival of patients with human breast cancer cells.更多还原