Neuropilin-1在食管癌中的表达调控及其作用机制研究

发布时间：2018-05-05 17:32

本文选题：食管鳞癌 + NRP1　；参考：《北京协和医学院》2015年博士论文

【摘要】：食管癌发生发展过程中存在大量分子异常改变。实验室前期工作中获得一个食管癌组织高表达蛋白Calreticulin(CRT),功能研究发现CRT可促进食管癌细胞侵袭迁移,表达谱芯片分析显示包括Neuropilin-1(NRP1)在内的多个基因在食管癌细胞中受CRT调控。本工作进一步研究了NRP1介导CRT影响食管癌细胞恶性表型的分子机制。Real-time PCR与Western blot验证结果表明CRT可以促进NRP1mRNA口蛋白表达。体外细胞侵袭与迁移实验显示,敲降NRP1食管癌细胞侵袭迁移能力明显降低；敲降CRT回复NRP1表达,食管癌细胞侵袭迁移能力得到回复。裸鼠肺转移实验显示,抑制NRP1表达,食管癌细胞肺转移结节数量显著降低。敲降CRT或NRP1均能抑制MMP2、MMP9及FAK的表达,提示CRT-NRP1通路异常可能是食管癌细胞侵袭迁移的重要机制。染色质免疫沉淀(ChIP)与双荧光素酶报告基因实验结果显示,转录因子STAT5A能够调控NRP1的转录。检测发现CRT增强STAT5A的转录活性。在食管癌组织中验证发现,CRT与NRP1表达显著正相关。这些结果说明,我们发现了一个新的促进食管癌细胞侵袭迁移信号通路,即CRT-STAT5A-NRP1。食管癌组织分析显示,NRP1高表达与原发性肿瘤浸润深度和患者不良预后显著正相关。细胞表型研究结果显示,NRP1可以增强食管癌细胞增殖及裸鼠成瘤能力。进一步研究发现,NRP1可以上调N F-κB P65亚基的mRNA和蛋白表达,回复实验结果提示NRP1可调控P65表达进而影响食管癌细胞的增殖。由于NRP1不具备转录因子功能,我们进一步研究了介导NRP1促进P65转录的机制。通过生物信息学分析、ChIP实验和双荧光素酶报告基因实验获得了P65的候选转录因子cAMP反应元件结合蛋白CREB。我们发现NRP1可与表皮生长因子受体EGFR相互作用并激活EGFR,NRP1的b1/b2结构域对EGFR的活化起到关键作用；EGFR通过AKT通路调控CREB的转录因子活性,进而影响P65表达。在食管癌组织中利用连续切片检测发现,NRP1与P65表达水平显著正相关。由此我们揭示了NRP1调控食管癌增殖的信号通路,即NRP1-EGFR-AKT-CREB-P65。利用NRP1靶向多肽处理肿瘤细胞,结果显示多肽终浓度为5μmol/L时,大多数NRP1高表达的肿瘤细胞系生长显著受到抑制；按照250μg/只的剂量,每周腹腔注射给药2次,能够明显抑制食管癌细胞裸鼠皮下瘤的生长,提示NRP1可能作为潜在的肿瘤治疗靶点。
[Abstract]:There are a lot of abnormal molecular changes during the development of esophageal carcinoma. A high expression protein Calreticulinine (CRT) was obtained in our laboratory. Functional studies showed that CRT could promote invasion and migration of esophageal cancer cells. Expression microarray analysis showed that many genes, including Neuropilin-1 NRP1, were regulated by CRT in esophageal cancer cells. In this work, we further studied the molecular mechanism of CRT mediated by NRP1 on the malignant phenotype of esophageal cancer cells. Real-time PCR and Western blot verification showed that CRT could promote the expression of NRP1mRNA protein. In vitro cell invasion and migration assay showed that knockdown of NRP1 esophageal carcinoma cells significantly decreased the ability of invasion and migration, knock down CRT returned to NRP1 expression, esophageal cancer cell invasion and migration ability was restored. Lung metastasis test in nude mice showed that inhibition of NRP1 expression significantly decreased the number of metastatic nodules in esophageal cancer cells. Knockdown of CRT or NRP1 can inhibit the expression of MMP2, MMP9 and FAK, suggesting that abnormal CRT-NRP1 pathway may be an important mechanism of invasion and migration of esophageal cancer cells. The results of chromatin immunoprecipitation and double luciferase reporter gene experiments showed that the transcription factor STAT5A could regulate the transcription of NRP1. CRT was found to enhance the transcriptional activity of STAT5A. A positive correlation between CRT and NRP1 expression was found in esophageal carcinoma tissues. These results suggest that we have discovered a new signaling pathway that promotes invasion and migration of esophageal cancer cells, namely CRT-STAT5A-NRP1. Tissue analysis showed that the high expression of NRP1 was positively correlated with the depth of primary tumor invasion and poor prognosis. The results of phenotypic study showed that NRP1 could enhance the proliferation of esophageal cancer cells and the tumorigenic ability of nude mice. Furthermore, it was found that NRP1 could up-regulate the expression of mRNA and protein of NF- 魏 B p65 subunit, and the response experiment suggested that NRP1 could regulate the expression of p65 and affect the proliferation of esophageal carcinoma cells. Since NRP1 does not have the function of transcription factor, we have further studied the mechanism of mediating NRP1 to promote the transcription of p65. The candidate transcription factor cAMP response element binding protein CREB for p65 was obtained by bioinformatics analysis and double luciferase reporter gene experiment. We found that NRP1 interacts with epidermal growth factor receptor EGFR and activates the b1/b2 domain of EGFR NRP1, which plays a key role in the activation of EGFR. EGFR regulates the activity of CREB transcription factors through the AKT pathway, thus affecting the expression of p65. A positive correlation between the expression of NRP1 and p65 was found in esophageal carcinoma by serial section detection. Therefore, we reveal the signal pathway of NRP1 regulating the proliferation of esophageal carcinoma, namely NRP1-EGFR-AKT-CREB-P65. Tumor cells were treated with NRP1 targeted polypeptides. The results showed that the growth of most tumor cell lines with high NRP1 expression was significantly inhibited when the final concentration of polypeptide was 5 渭 mol/L, and was injected twice a week at the dose of 250 渭 g / mouse. It can obviously inhibit the growth of subcutaneous tumor of esophageal cancer cells in nude mice, suggesting that NRP1 may be a potential target for tumor therapy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.1

【共引文献】