酪氨酸代谢酶 HPD 在肺癌发生和发展中的作用及机制研宄

发布时间：2019-03-02 16:32

【摘要】：本论文的研究内容分为两部分,第一部分对酪氨酸代谢酶HPD(羟苯丙酮酸二加氧酶)在肺癌发生和发展中的作用及作用机制进行了系统研究,第二部分在前期研究基础上,深入探讨了转录因子THAP11在造血分化中的作用机制。HPD是酪氨酸代谢中的一个关键酶,以往的研究表明HPD在肝脏及肾脏中特异表达,在其他组织中表达水平低或不表达。我们利用组织芯片检测了HPD在355例肺癌病人中的表达水平,结果显示HPD在肺癌组织中的表达水平显著高于癌旁组织,进一步利用ELISA和Western Blotting技术验证了HPD在肺癌组织和细胞系的高表达。为探讨HPD异常高表达对肺癌细胞恶性表型的影响,我们构建了基于慢病毒的HPD过表达及HPD RNAi的肺癌稳定细胞株,研究发现过表达HPD并不影响肺癌细胞的生长,但敲低HPD则显著抑制肺癌细胞的生长与存活,并降低肺癌细胞体内成瘤能力,表明HPD参与肺癌细胞生长和存活能力的调控。此外,过表达HPD促进多种肺癌细胞的迁移能力,敲低HPD则抑制肺癌细胞的迁移能力,并降低肺癌细胞在体内的转移能力,表明HPD过表达促进肺癌细胞侵袭转移。我们还检测了HPD对治疗肺癌药物敏感性的影响,结果显示过表达HPD降低肺癌细胞对多种药物的敏感性,而敲低HPD可导致肺癌细胞对药物的敏感性增加,凋亡增多。另外,我们对HPD的作用机制进行初步探讨,发现HPD的酶活性特异抑制剂Nitisinone对肺癌细胞增殖和迁移无明显影响,提示HPD对肺癌细胞的影响并不依赖其代谢酶活性。根据前期研究基础,我们发现HPD促进肺癌细胞中NF-?B信号通路的活化,表现为HPD增加NF-кB报告基因活性,促进IкBα降解及p65入核,上调NF-кB下游靶基因如c-Myc、MMP9和CyclinD1等的表达,表明HPD可能通过促进NF-кB信号通路调控肺癌细胞存活、凋亡和迁移。THAP11是一种转录因子,可通过结合c-Myc启动子下调c-Myc表达抑制细胞增殖,在增殖、胚胎发育及ES细胞分化过程中发挥重要作用。我们前期的研究表明THAP11是一个新的调控红系/巨核系分化的转录因子,在调控造血细胞向红系和巨核系分化过程中发挥着重要的作用,但调控机制并不明确。我们对THAP11在造血分化中的作用机制进行深入研究,结果显示在hemin诱导K562细胞向红系分化过程中,THAP11过表达可导致红系造血因子c-Myc下调,巨核系造血因子Fli 1上调。THAP11在多种造血转录因子如GATA-2、c-Myb、Fli-1和c-Myc等的启动子区存在结合位点,ChIP实验证实THAP11与这些位点存在结合。在PMA诱导K562细胞向巨核系分化过程中,THAP11并不影响H3K9和H3K18的乙酰化水平。此外,我们还发现THAP11和GATA-2存在相互作用,相互作用结构域在GATA-2的锌指结构区,提示THAP11很可能通过相互作用调控GATA-2活性。这些研究结果表明THAP11参与造血分化可能是红系或巨核系相关基因进行整体调控的结果。
[Abstract]:The research contents of this thesis are divided into two parts. In the first part, the role and mechanism of tyrosine metabolizing enzyme HPD (hydroxyphenylketoate dioxygenase) in the carcinogenesis and development of lung cancer are studied systematically. The second part is based on the previous study. The mechanism of transcription factor THAP11 in hematopoiesis differentiation was investigated. HPD is a key enzyme in tyrosine metabolism. Previous studies have shown that HPD is specifically expressed in liver and kidney, but not expressed in other tissues. We detected the expression of HPD in 355 patients with lung cancer using tissue microarray. The results showed that the expression level of HPD in lung cancer tissues was significantly higher than that in non-cancerous tissues. Furthermore, ELISA and Western Blotting techniques were used to verify the high expression of HPD in lung cancer tissues and cell lines. In order to investigate the effect of abnormal high expression of HPD on malignant phenotype of lung cancer cells, we constructed a stable lung cancer cell line based on lentiviral HPD overexpression and HPD RNAi. It was found that overexpression of HPD did not affect the growth of lung cancer cells. However, knock-down of HPD significantly inhibited the growth and survival of lung cancer cells, and decreased the tumorigenicity of lung cancer cells in vivo, suggesting that HPD is involved in the regulation of growth and survival of lung cancer cells. In addition, over-expression of HPD promoted the migration of lung cancer cells, while down-regulation of HPD inhibited the migration of lung cancer cells and decreased the metastatic ability of lung cancer cells in vivo, indicating that over-expression of HPD promoted the invasion and metastasis of lung cancer cells. We also examined the effect of HPD on the drug sensitivity of lung cancer cells. The results showed that over-expression of HPD decreased the sensitivity of lung cancer cells to many kinds of drugs, and low HPD resulted in increased drug sensitivity and increased apoptosis of lung cancer cells. In addition, we investigated the mechanism of HPD and found that Nitisinone, a specific inhibitor of HPD enzyme activity, had no significant effect on the proliferation and migration of lung cancer cells, suggesting that the effect of HPD on lung cancer cells did not depend on its metabolic enzyme activity. On the basis of previous studies, we found that HPD promoted the activation of NF-?B signaling pathway in lung cancer cells, which showed that HPD increased the activity of NF- B reporter gene, promoted the degradation of I / B 伪 and the entry of p65 into the nucleus, and up-regulated the downstream target genes of NF- B, such as ccMyc. The expression of MMP9 and CyclinD1 suggests that HPD may regulate the survival, apoptosis and migration of lung cancer cells by promoting NF- B signaling pathway. THAP11 is a transcription factor, which can inhibit the proliferation of lung cancer cells by down-regulating the expression of c-Myc by binding to c-Myc promoter. Embryonic development and differentiation of ES cells play an important role. Our previous studies have shown that THAP11 is a new transcription factor regulating erythroid / megakaryocyte differentiation, which plays an important role in regulating the differentiation of hematopoietic cells into erythroid and megakaryocyte lines, but the regulatory mechanism is not clear. We studied the mechanism of THAP11 in hematopoiesis differentiation. The results showed that the overexpression of THAP11 could lead to down-regulation of erythroid hemopoietic factor c-Myc during the differentiation of K562 cells to erythroid induced by hemin. Megakaryocyte hemopoietic factor Fli-1 was up-regulated. THAP11 had binding sites in the promoter regions of various hematopoietic transcription factors, such as GATA-2,c-Myb,Fli-1 and c-Myc, and THAP11 binding to these sites was confirmed by ChIP assay. During the differentiation of K562 cells into megakaryocytes induced by PMA, THAP11 did not affect the acetylation level of H3K9 and H3K18. In addition, we also found that THAP11 and GATA-2 interact with each other, and the interaction domain is in the zinc finger domain of GATA-2, suggesting that THAP11 may regulate the activity of GATA-2 through the interaction. These results suggest that the involvement of THAP11 in hematopoietic differentiation may be the result of the whole regulation of erythroid or megakaryocyte-related genes.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2

【相似文献】