突变p53诱导转录因子Egr-1调控Cathepsin L在肺癌上皮间质转化中的作用

发布时间：2018-05-21 04:15

本文选题：Cathepsin + L　；参考：《苏州大学》2016年博士论文

【摘要】：第一部分突变p53与Cathepsin L表达特征的关系以及在肺癌患者上皮间质转化中的作用目的:探究78例非小细胞肺癌患者肿瘤组织中p53基因的突变情况与Cathepsin L表达的特征以及EMT相关蛋白表达的相关性,并分析它们的表达变化与临床病理的关系。方法:收集78例人肺癌及其癌旁组织标本,对肿瘤组织进行p53基因突变检测,分析其突变规律与肺癌病理特征的关系;同时采用Western blot、ELISA及免疫组织化学检测Cathepsin L蛋白的表达情况,分析其与肺癌病理特征的关系;采用Western blot和免疫组织化学检测肺癌组织中上皮间质转化标志物E-cadherin和N-cadherin的表达,分析p53基因突变、Cathepsin L与上皮间质转化的相关性。结果:肺癌组织中p53基因突变率为56%,大多为GC→AT和GC→TA的碱基颠换;p53基因突变与患者的分化程度密切相关。与正常肺组织相比,活性Cathepsin L在肿瘤组织中表达明显上调;无论在肿瘤细胞中还是病人的血清样本里,Cathepsin L的表达与肺癌组织的级别密切相关。p53基因突变的肺癌组织中Egr-1激活,Cathepsin L入核现象明显,而且EMT相关蛋白表达发生相应的变化。结论:突变p53肺癌组织中EMT通路被激活;Cathepsin L的过度表达参与肺癌的发生发展,可以作为肺癌患者预后的指标。第二部分突变p53诱导Cathepsin L在肺癌细胞上皮间质转化中的调控机制目的:研究野生型p53基因和突变型p53基因诱导Cathepsin L影响上皮间质转化的调控机制,以此探讨突变p53基因获得性功能在人肺癌侵袭迁移中的作用。方法:体外构建稳定感染不同p53基因状态慢病毒的细胞株,用电离辐射处理细胞,采用划痕法、Transwell小室法分别检测肿瘤细胞的迁移、侵袭能力;鬼笔环肽染色的方法检测肿瘤细胞骨架变化;Western blot及免疫荧光检测上皮间质转化相关标志蛋白的变化;染色质免疫共沉淀法检测p53和Egr-1参与调节下游靶基因Cathepsin L的转录激活水平;建立裸小鼠皮下移植瘤模型,观察不同p53基因状态的细胞株在电离辐射治疗后的成瘤能力,运用Western blot、免疫组化分析法检测各组移植瘤Cathepsin L与上皮间质转化相关标志蛋白的表达情况。结果:在辐照诱导后,稳定感染p53基因突变慢病毒的细胞株Cathepsin L和Egr-1表达上调,并且侵袭和迁移能力得到了提高;ChIP结果显示突变型p53对Cathepsin L的转录过程是依赖Egr-1的。在裸小鼠皮下移植瘤模型中,突变组的放疗效果却没有野生组理想;免疫组化分析显示,相较于野生组细胞株,突变组细胞株的移植瘤中Cathepsin L定位在细胞核,E-cadherin的表达减少,而N-cadherin的表达则增加,与细胞实验结果一致。结论:突变p53能通过诱导转录因子Egr-1调控Cathepsin L的表达,继而促进肿瘤细胞的上皮间质转化,影响肿瘤细胞的侵袭迁移能力。突变型p53和Egr-1协同参与对Cathepsin L的转录过程,提示p53基因状态和Cathepsin L蛋白在调控肿瘤细胞侵袭迁移方面起着重要的作用。本研究结果将为Cathepsin L成为肿瘤治疗的有效靶点提供实验依据。
[Abstract]:The relationship between the first part of the mutation p53 and the expression of Cathepsin L and the role in the transformation of epithelial mesenchymal transition in lung cancer patients: To explore the mutation of the p53 gene in the tumor tissues of 78 patients with non-small cell lung cancer and the characteristics of the expression of Cathepsin L and the correlation of the expression of EMT related proteins, and to analyze their expression changes and clinical diseases. Methods: 78 cases of human lung cancer and its adjacent tissue specimens were collected, and the p53 gene mutation was detected in the tumor tissue. The relationship between the mutation law and the pathological features of lung cancer was analyzed. The expression of Cathepsin L protein was detected by Western blot, ELISA and immunohistochemistry, and the relationship between the pathological features of the lung cancer and the pathological features of lung cancer was analyzed, and Wes was used in Wes. The expression of epithelial mesenchymal transition markers E-cadherin and N-cadherin in lung cancer tissues was detected by tern blot and immunohistochemistry. The correlation between p53 gene mutation, Cathepsin L and epithelial mesenchymal transition was analyzed. Results: the mutation rate of p53 gene in lung cancer tissues was 56%, most of which were GC, AT and GC to TA. The expression of the active Cathepsin L in the tumor tissue was obviously up-regulated compared with the normal lung tissue. In the tumor cells or in the patient's serum samples, the expression of Cathepsin L was closely related to the activation of Egr-1 in the lung cancer tissues of the.P53 gene mutation, and the Cathepsin L had a obvious nucleation, and EMT. Corresponding changes in the expression of related proteins. Conclusion: the EMT pathway in the mutant p53 lung cancer is activated; the overexpression of Cathepsin L is involved in the development of lung cancer, and it can be used as an indicator of prognosis of lung cancer patients. Second partial mutation p53 induces the mechanism of Cathepsin L in the transformation of epithelial mesenchymal transition of lung cancer cells: the study of wild type p53 The gene and mutant p53 genes induce Cathepsin L to influence the regulatory mechanism of epithelial mesenchymal transition, in order to explore the role of the mutant p53 gene in the invasion and migration of human lung cancer. Methods: to construct a cell line with lentivirus infection of different p53 genes in vitro, use ionizing radiation to treat cells, use scratch method, Transwell chamber method. Detection of tumor cell migration, invasiveness, cytoskeleton changes of tumor cells, Western blot and immunofluorescence detection of epithelial mesenchymal transition related markers; chromatin immunoprecipitation assay p53 and Egr-1 to regulate the transcriptional activation level of Cathepsin L of the downstream target gene; establish nude The mouse subcutaneous transplantation tumor model was used to observe the tumorigenicity of the cell lines with different p53 gene States after ionizing radiation. The expression of Cathepsin L and epithelial mesenchymal transition related markers was detected by Western blot and immunohistochemistry. Results: after irradiated, the p53 gene mutated to the lentivirus. The expression of Cathepsin L and Egr-1 was up-regulated, and the ability to invasion and migration was improved; ChIP results showed that the transcriptional process of mutant p53 to Cathepsin L was Egr-1. In the nude mice model, the radiotherapy effect of the mutant group was not ideal for the wild group; immunohistochemical analysis showed that it was compared to the wild group cell line. In the transplanted tumor of the mutant group, Cathepsin L was located in the nucleus, the expression of E-cadherin decreased, while the expression of N-cadherin increased, which was in agreement with the results of cell experiment. Conclusion: mutation p53 can regulate the expression of Cathepsin L by inducing the transcription factor Egr-1, and then promote the epithelial mesenchymal transformation of the tumor cells and affect the invasion of the tumor cells. Migration ability. Mutational p53 and Egr-1 participate in the transcriptional process of Cathepsin L, suggesting that p53 gene state and Cathepsin L protein play an important role in regulating tumor cell invasion and migration. The results of this study will provide a practical basis for Cathepsin L to be an effective target for cancer treatment.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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