ATF-2的表达对非小细胞肺癌细胞增殖和不良预后的影响

发布时间：2017-12-28 03:01

本文关键词：ATF-2的表达对非小细胞肺癌细胞增殖和不良预后的影响　出处：《南昌大学》2017年博士论文　论文类型：学位论文

【摘要】：非小细胞肺癌是全世界最常见的癌症,全球每年新增病例超120万。激活转录因子-2(activating transcription factor 2,ATF-2)属于转录因子ATF家族蛋白,是转录复合体的不可缺少的组成部分。在细胞核或细胞质ATF2的亚细胞定位不同,呈现出双功能活性致癌或抑癌基因,ATF2的核定位和其致癌功能相关,细胞质定位与其发挥肿瘤抑制功能相关。本研究主要探讨非小细胞肺癌组织中ATF2表达及临床分期、无病生存率的关联情况,同时研究了ATF2在人类非小细胞肺癌细胞系的表达与细胞增殖的相关性,并初步确定ATF2可以成为非小细胞肺癌预后的潜在预测标志物。目的:探讨ATF-2蛋白表达水平对非小细胞肺癌细胞增殖和预后的影响。方法:1、采集86例非小细胞肺癌患者的病理切片,免疫组化检测肺癌组织及癌旁组织ATF-2和pATF-2蛋白的表达情况,并统计分析ATF-2与患者临床病理学参数的相关性。2、采集10例非小细胞肺癌术后新鲜肺癌组织和癌旁组织标本,通过qRT-PCR、Western Blot等方法检测其ATF-2 mRNA和ATF-2蛋白的表达水平。3、shRNA转染H1975和H1299肺癌细胞系,使其干扰ATF-2基因表达,转染48h后Western Blot法检测肺癌细胞系的ATF-2蛋白表达,并检测H1975和H1299的增殖活性变化。4、通过质粒转染技术构建稳定表达ATF-2蛋白的HBE细胞株,并采用集落形成试验和WST-8试验检测其增殖活性的变化。结果:1.ATF2 mRNA在肿瘤组织比癌旁组织有明显上调,肿瘤的ATF2蛋白质含量高于癌旁组织。2.ATF2表达与非小细胞肺癌临床分期有显著相关性,ATF2表达水平越高,分期越晚。3.ATF2和pATF2在淋巴结转移的非小细胞癌中的标本和没有伴随淋巴转移的标本组织中阳性表达差别是有显著性,ATF2和pATF2的高表达是与淋巴结的转移有密切关系的。4.在非小细胞肺癌标本中存在ATF2高表达的患者,其无病生存时间显著缩短,ATF2低表达和较长的无病生存时间相关,高p-ATF2表达和生存时间缩短呈显著相关,和无病生存时间存在极显著相关。5.ATF2和pATF2表达与患者的年龄、性别、组织大小无显著相关性。6.pATF2表达主要出现在非小细胞肺癌的细胞核中。细胞质也能够观察到分散染色的p-ATF2的表达。在86例患者肿瘤组织中,有65例属于pATF2高表达(75.6%)。在细胞质和细胞核中,ATF2的表达没有显著性差异。肿瘤癌旁正常组织中,ATF2和pATF2的表达没有显著性差异(P=0.056)。ATF2对于非小细胞肺癌的预后被观测到有预测意义,ATF2阳性表达和临床病理因素如年龄,性别和转移之间的相关性没有统计学意义。NSCLC患者细胞核高表达pATF2与肿瘤晚期,肿瘤大小和转移具有显著相关性。7.ATF2在非小细胞肺癌细胞株表达阳性,在细胞系H446、H1975H1650,H1299,PC9的阳性表达明显增高。8.ATF2的siRNA被证实能够有效的对ATF2的表达进行下调,利用MTT实验分析,显示在H1299和H1975细胞中,低表达的ATF2能够导致细胞生存能力的降低,ATF2的阳性表达能够促进细胞增殖,而下调ATF2则能够抑制细胞增殖。9.在HBE细胞株中,ATF2表达水平升高可以促进HBE细胞的增殖生长。结论:ATF2在非小细胞肺癌组织中存在过表达,并与患者的预后存在一定的相关性,有可能成为潜在的预后指标。体外实验中,ATF2表达上调可以促进肺癌细胞系的增殖,而下调ATF2表达后肺癌细胞系的增殖能力明显减弱。该研究初步提示ATF2有望成为非小细胞肺癌新的预后因素,尚需进一步的深入研究。
[Abstract]:Non - small cell lung cancer (non-small cell lung cancer) is the most common cancer in the world, with more than 1 million 200 thousand new cases in the world each year. The activation of transcription factor -2 (activating transcription factor 2, ATF-2) belongs to the transcription factor ATF family protein, which is an indispensable component of the transcription complex. The subcellular location of ATF2 in nuclei or cytoplasm is different, showing a bifunctional active carcinogen or tumor suppressor gene. ATF2's nuclear localization is related to its carcinogenesis function. Cytoplasmic localization is related to its tumor inhibition function. This study focuses on non small cell lung cancer ATF2 expression and clinical stage, no correlation between survival rate, and study the correlation between the expression of ATF2 in human NSCLC cell lines and cell proliferation, and determined ATF2 can become a potential predictive biomarkers of non small cell lung cancer prognosis. Objective: To investigate the effect of ATF-2 protein expression on the proliferation and prognosis of non-small cell lung cancer cells. Methods: 1. 86 cases of non-small cell lung cancer were collected for pathological sections. Immunohistochemistry was used to detect the expression of ATF-2 and pATF-2 protein in lung cancer tissues and adjacent tissues. The correlation between ATF-2 and clinicopathological parameters was analyzed statistically. 2. We collected 10 cases of fresh lung cancer and adjacent tissues after operation, and detected the expression level of ATF-2 mRNA and ATF-2 protein by qRT-PCR and Western Blot. 3, shRNA transfected H1975 and H1299 lung cancer cell lines to interfere with ATF-2 gene expression. After transfection of 48h, Western ATF-2 method was used to detect ATF-2 protein expression in lung cancer cell lines, and the proliferation activity of H1975 and H1299 was detected. 4, HBE cell lines expressing ATF-2 protein were constructed by plasmid transfection technique, and colony-forming test and WST-8 test were used to detect the changes of proliferation activity. Results: 1.ATF2 mRNA was significantly up-regulated in tumor tissue than that of para cancer tissue, and the content of ATF2 protein in tumor was higher than that of para cancerous tissue. There is a significant correlation between the expression of 2.ATF2 and the clinical stage of non-small cell lung cancer. The higher the expression of ATF2, the later the stage is. The positive expression of 3.ATF2 and pATF2 in non small cell carcinoma with lymph node metastasis and those without lymph node metastasis is significant. The high expression of ATF2 and pATF2 is closely related to lymph node metastasis. 4. in patients with non-small cell lung cancer, patients with high expression of ATF2 had significantly shorter disease-free survival time, lower ATF2 expression and longer disease-free survival time. High p-ATF2 expression was significantly correlated with shorter survival time, and had significant correlation with disease-free survival time. There was no significant correlation between the expression of 5.ATF2 and pATF2 and the age, sex and size of the patients. The expression of 6.pATF2 mainly occurs in the nucleus of non small cell lung cancer. The cytoplasm can also be used to observe the expression of the dispersed p-ATF2. Of the 86 patients with tumor tissue, 65 were of high expression of pATF2 (75.6%). There was no significant difference in the expression of ATF2 in the cytoplasm and nucleus. There was no significant difference in the expression of ATF2 and pATF2 (P=0.056) in the normal tissues adjacent to the tumor. ATF2 has a predictive significance for the prognosis of non-small cell lung cancer. There is no significant correlation between ATF2 positive expression and clinicopathological factors, such as age, gender and metastasis. The high expression of pATF2 in the cell nucleus of NSCLC has a significant correlation with the advanced tumor, tumor size and metastasis. The expression of 7.ATF2 was positive in non small cell lung cancer cell lines, and the positive expression of H446, H1975H1650, H1299 and PC9 in the cell lines was significantly higher. 8.ATF2 siRNA proved to be effective on the expression of ATF2 was down regulated by MTT analysis of the experimental results, shown in H1299 and H1975 cells, low expression of ATF2 can reduce the lead to cell viability, the positive expression of ATF2 can promote cell proliferation and downregulation of ATF2 can inhibit cell proliferation. 9. in the HBE cell line, the increase of ATF2 expression level can promote the proliferation and growth of HBE cells. Conclusion: ATF2 is overexpressed in non-small cell lung cancer and has a certain correlation with the prognosis of the patients, and it may be a potential prognostic indicator. In vitro, the up-regulated expression of ATF2 could promote the proliferation of lung cancer cell lines, and the proliferation ability of lung cancer cell lines decreased significantly after the expression of ATF2 was downregulated. This study suggests that ATF2 is expected to be a new prognostic factor for non-small cell lung cancer and needs further in-depth study.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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