【作者】 郭伟；

【导师】 蒋耀光；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 背景食管癌是目前对人类健康和生命构成严重威胁的恶性肿瘤之一,在各种恶性肿瘤导致的死亡中食管癌占第六位,在我国一些高发地区居于各种恶性肿瘤的首位。食管癌预后较差,主要原因是肿瘤在早期即可发生外侵或转移。食管癌的发生发展是多基因共同作用的后果。目前在食管癌组织中已发现多种表达异常的基因和蛋白,但具有临床应用价值的分子标志物却甚少,能够对食管癌患者治疗效果和预后判断提供可靠依据的标志物尚需进一步研究证实和寻找。锌带蛋白基因1(Zinc ribbon domain-containing 1,ZNRD1)是一种与转录相关的锌带蛋白,可能通过调节转录起始位点的选择、RNA合成的延长和终止在转录中发挥重要调控作用。ZNRD1的表达可以抑制胃癌细胞系在体内外的生长和致瘤性,并且能够抑制胃癌细胞系体外转化表型,进而逆转一些胃癌细胞系在体内外的恶性生长潜能,表明ZNRD1在肿瘤发展中能发挥一定的抑制作用,有可能是潜在的肿瘤基因治疗靶点。ZNRD1在食管癌细胞内的作用、其抗凋亡的机制、与食管癌发生有无关系等均未见报道。本研究一方面旨在探讨ZNRD1在食管癌组织中的差异表达,了解ZNRD1表达水平与患者临床病理特征和预后之间的关系;另外,以ZNRD1真核正义表达载体转染人源性食管鳞癌细胞系EC109,构建ZNRD1基因高表达的食管鳞癌细胞亚系,探讨ZNRD1过表达对EC109细胞增殖、凋亡、转移以及DNA损伤修复等生物学行为的影响和作用机制,为深入理解ZNRD1在食管癌中的作用奠定理论基础。研究结果可望为食管癌的基因治疗和预后判断提供具有一定临床应用价值的选择。目的研究ZNRD1基因在食管癌组织及其对应近癌、远癌组织中的表达情况,了解ZNRD1表达水平与患者临床病理特征和预后之间的关系,为ZNRD1作为食管癌预后判断候选基因提供实验依据;检测食管鳞癌细胞系EC109中ZNRD1表达水平,构建ZNRD1表达上调的食管鳞癌细胞亚系,研究ZNRD1表达对食管鳞癌细胞系EC109增殖、凋亡以及转移等生物学行为的影响和作用机制;探讨ZNRD1表达水平对EC109细胞化疗药物敏感性及DNA损伤修复功能的影响及其可能的分子机制。方法(1)应用逆转录聚合酶链反应(RT-PCR)和免疫组织化学(IHC)技术检测食管癌患者肿瘤组织及其对应近癌、远癌组织中ZNRD1基因的表达;(2)采用IHC技术检测食管癌患者肿瘤组织中ZNRD1蛋白的表达,结合随访资料进行统计分析;(3)应用RT-PCR、Western blot和免疫细胞化学(ICC)技术检测ZNRD1基因在食管鳞癌细胞系EC109中的表达;(4)利用脂质体转染法建立ZNRD1基因过表达的食管鳞癌细胞模型,并利用RT-PCR和Western blot技术进行鉴定;(5)利用MTT实验绘制转染细胞、对照细胞及亲本细胞的生长曲线;(6)通过Transwell侵袭小室实验检测ZNRD1过表达对食管鳞癌细胞侵袭活性的影响;(7)通过流式细胞仪分析转染细胞及对照细胞的细胞周期和细胞凋亡的变化;(8)通过MTT实验进行体外药物敏感性分析,计算细胞对药物的IC50值;(9)通过流式细胞仪和DNA断裂实验检测ZNRD1过表达对EC109细胞凋亡敏感性的影响;(10)利用单细胞凝胶电泳(SCGE)技术检测紫外线(UV)照射对转染细胞及其对照细胞的DNA损伤修复效应;(11)通过基因芯片检测ZNRD1过表达对EC109细胞基因表达谱的影响;(12)利用RT-PCR和Western blot对基因芯片的结果进行鉴定。结果(1) ZNRD1在食管癌组织中的表达水平明显低于近癌及远癌组织;ZNRD1在食管癌患者肿瘤组织及其对应近癌、远癌组织中的表达率分别为29.5%、52.3%和72.7%,ZNRD1在近癌及远癌组织中的表达率显著高于肿瘤组织(p < 0.05);(2)转染细胞经RT-PCR和Western blot证实,成功建立了ZNRD1过表达的食管鳞癌细胞模型;(4) MTT实验及集落形成实验结果表明,上调ZNRD1可以导致食管鳞癌细胞系EC109生长及增殖速度减慢;(5) Transwell侵袭小室实验结果显示,上调ZNRD1可以显著降低食管鳞癌细胞系EC109的侵袭活性;(6)流式细胞仪检测结果表明,上调ZNRD1可以导致EC109细胞出现G1期阻滞,S期比例减少;(7)体外药物敏感实验结果表明,上调ZNRD1能够显著降低EC109细胞对5-氟尿嘧啶(5-Fu)、顺铂(CDDP)、长春新碱(VCR)和阿霉素(ADR)的敏感性,增强EC109细胞对化疗药物的耐受性;(8)流式细胞仪检测及DNA断裂实验结果表明,上调ZNRD1可以降低EC109细胞对阿霉素的敏感性,增加食管鳞癌细胞株EC109的抗凋亡能力;(9) SCGE实验结果表明,上调ZNRD1表达水平能够显著增强EC109细胞的DNA修复能力,抑制紫外线照射对食管鳞癌细胞EC109的DNA损伤效应;(10)基因芯片检测结果表明,上调ZNRD1可能改变61种基因的表达水平;(11)经RT-PCR和Western blot证实,转染ZNRD1可以显著上调ERCC1、P-gp、Bcl-2、和MDR1等基因的表达。结论ZNRD1在食管癌组织中的表达水平显著低于对应近癌及远癌组织;ZNRD1表达水平与患者预后呈正相关:肿瘤组织中ZNRD1表达水平高者其预后明显优于ZNRD1低表达患者。食管鳞癌细胞系EC109中ZNRD1 mRNA及蛋白表达均为阴性,上调ZNRD1可以在一定程度上逆转食管鳞癌细胞系EC109的恶性表型,导致EC109细胞生长和增殖速度明显减慢,细胞出现G1期阻滞,抑制EC109细胞的侵袭活性,ZNRD1可能通过调节CDKN1A等基因表达参与细胞周期调控。上调ZNRD1可以降低EC109细胞的药物敏感性,抑制EC109细胞凋亡。ZNRD1可能通过调节DNA损伤修复相关基因ERCC1及Bcl-2的表达水平进而参与紫外线及铂类化疗药物等DNA损伤因子对EC109细胞的DNA损伤效应。更多还原

【Abstract】 BackgroundEsophageal cancer is one of the most prevalent and deadliest malignancies. Worldwide, esophageal cancer is the sixth leading cause of death from cancer, and even the most frequent malignant tumor in several regions of China. Due to metastasis and invasion of surrounding tissues in early stage, the prognosis of esophageal cancer still remains poor. Numerous genes involve in the pathogenesis of esophageal cancer, but the concrete process remains unclear. Multiple genes and proteins with abnormal expression have been found in esophageal cancer. However, a few of them can be adopted as biomarkers with clinical value. The other biomarkers related to evaluating therapeutic efficacy and prognosis need further searching and confirming. As a zinc band protein related to transcription, the ZNRD1 (Zinc ribbon domain-containing 1) gene might play important role in the process of transcription through regulating the choice of transcriptional start site, as well as the extension and termination for synthesis of RNA. Overexpression of ZNRD1 can suppress the proliferation and oncogenicity of stomach cancer cells in vitro and in vivo, and restrain its transform phenotype, thus reversing the malignant proliferation potency of some stomach cancer cells. These fingings suggest that ZNRD1 gene transduction may be a useful new strategy of gene therapy for gastric cancer. However, there is still no report about the role of ZNRD1 in esophageal cancer cells, and the precise mechanism of apoptosis regulation by ZNRD1, as well as the clinical value of ZNRD1 in evaluating therapeutic efficacy and prognosis. In this study, we explored the differential expression of ZNRD1 in esophageal cancer tissues; to understanded the relation between the level of ZNRD1 expression and clinical pathological features, as well as the prognosis of patients underwent radical esophagectomy. In addition, we transfected the eukaryotic sence expression vector of ZNRD1 into EC109 cells, and also explored the function and possible mechanisms of ZNRD1 related biological behaviours, such as proliferation, apoptosis, metastasis, as well as DNA damage and repair. All these laid a solid basis for the role of ZNRD1 in esophageal cancer.Aims: To identify the differential expression of ZNRD1 in esophageal cancer tissues and investigate the relationship between the expression of ZNRD1 and the prognosis of patients with esophageal cancer. In addition, the correlation between ZNRD1 expression and the clinical pathological characteristics was also investigated. For exploring the function and possible mechanisms of ZNRD1 related biological behaviours, such as proliferation, apoptosis, metastasis, as well as DNA damage and repair, the eukaryotic sence expression vector of ZNRD1 was transfected into EC109 cells.Methods: (1) Semiquantitative RT-PCR assay and immunohistochemistry staining technique were used to identify the differential expression of ZNRD1 mRNA and protein in esophageal cancer, normal and adjacent tissues.(2) The relationship between ZNRD1 expression and prognosis of patients with esophageal cancer was investigated by immunohistochemistry staining technique and statistical analysis.(3) The Western blot, RT-PCR assay and immunocytochemistry staining were used to detect the ZNRD1 expression in EC109 cells.(4) The eukaryotic sence expression vector of ZNRD1 was transfected into EC109 cells by liposome transfection technique, and then Western blot, RT-PCR assay and immunocytochemistry staining were used to detect the expression of ZNRD1.(5) Growth curve and drug sensitivity assay were performed using MTT assay. IC50 values of EC109 cells to anticancer drugs were calculated.(6) Invasion activity of transfected EC109 cells was investigated by Transwell loculus assay.(7) Cell cycle analysis was performed using flow cytometry.(8) The apoptosis sensitivity of transfectant and control cells was detected by DNA fragmentation assay and flow cytometry.(9) Utilizing single cell gel electrophoresis (SCGE) assay to evaluate the DNA damage and repair induced by ultraviolet light radiation.(10) The cDNA microarray analysis was performed to identify the gene expression profile mediated by ZNRD1 overexpression.(11) RT-PCR and Western blots were performed to validate the differential expression of genes identified by cDNA microarray analysis.Results: (1) the ZNRD1 expression was confirmed to be down-regulated in esophageal cancer tissues compared to normal and adjacent nonneoplastic tissues. As immunohistochemistry discovered, the positive expressing rate of ZNRD1 in esophageal cancer (29.5%) was lower than in adjacent nonneoplastic (52.3%) and normal tissues (72.7%) (p < 0.05).(2) The mRNA and protein levels of ZNRD1 were both undetectable in EC109 cells, while significantly higher expression of ZNRD1 mRNA and protein (p < 0.01) was observed in EC109/ZNRD1 cells. The results showed that ZNRD1 gene was successfully transfected into and expressed in the EC109 cells.(3) MTT and colony formation assay showed that upregulation of ZNRD1 could suppress cell growth and proliferation of EC109 cells.(4) As Transwell loculus assay showed, the invasion activity of EC109 cells was significantly suppressed by the overexpression of ZNRD1.(5) As showed by flow cytometry, ZNRD1 upregulated esophageal cancer cells EC109/ZNRD1 exhibited G1 stage blockage and less percentage of cells in S stage.(6) MTT assay showed that transfectant cell was more resistant to 5-Fu, CDDP, VCR and ADR than control cells.(7) According to the results of flow cytometry and DNA fragmentation assay, the apoptosis sensitivity of EC109/ZNRD1 was lower than control cells.(8) ZNRD1-expressing cells exhibited a significant enhanced DNA repair capacity, suggesting that upregulation of ZNRD1 could inhibit the DNA damage induced by ultraviolet light radiation.(9) As the cDNA microarray analysis discovered, there were 61 genes with different expression levels following the upregulation of ZNRD1 in EC109 cells.(10) Western blot showed that the overexpression of ZNRD1 can effectively upregulate the mRNA level of ERCC1, P-gp, MDR1 and Bcl-2 in EC109/ZNRD1, as compared with the control cells.Conclusions: ZNRD1 expression was confirmed to be down-regulated in esophageal cancer tissues compared to normal and adjacent nonneoplastic tissues by RT-PCR and immunohistochemistry. The expression of ZNRD1 in esophageal cancer was positive correlated to the prognosis of patients. The decreased expression of ZNRD1 was correlated with a worse outcome of patients with esophageal cancer. The mRNA and protein levels of ZNRD1 were both undetectable in EC109 cells, while significantly higher expression of ZNRD1 mRNA and protein was observed in EC109/ZNRD1 cells, which suggested that ZNRD1 gene was successfully transfected into and expressed in the EC109 cells. Upregulation of the ZNRD1 could partly reverse the malignant phenotype of EC109 cells, resulting in the suppressed cell growth, G1 stage blockage and the inhibited invasion activity. ZNRD1 might regulate the cell cycle by altering the expression of CDKN1A (Cyclin-dependent kinase inhibitor 1A). Moreover, upregulation of ZNRD1 could enhance the resistance of EC109 cells to chemotherapeutics and suppress the apoptosis. ZNRD1-expressing EC109 cells exhibited a significant enhanced DNA repair capacity, and the overexpression of ZNRD1 could upregulate the expression of excision repair cross-complementing 1 (ERCC1) gene. Thus suggested ZNRD1 might play an important role in the process of DNA damage and repair by regulating the expression of ERCC1. In addition, the ZNRD1 gene might be involved in the cisplatin resistance of EC109 cells by regulating the expression of ERCC1 and Bcl-2.更多还原