Fyn通过hnRNP E1调节integrin β1选择性剪接调控胰腺癌转移分子机制研究

发布时间：2018-04-12 17:47

本文选题：胰腺癌 + Fyn　；参考：《第三军医大学》2016年博士论文

【摘要】：一、研究背景胰腺癌是一种致死率极高的消化道恶性疾病,发病早期肿瘤细胞即发生局部侵袭与远处转移。胰腺癌的主要临床病理特征之一就是肿瘤细胞周围存在着大量的纤维组织以及丰富的细胞外基质成分(extracellular matrix,ECM)。这些特殊的ECM成分能够通过激活肿瘤细胞表面整合素Integrin及其胞内信号分子的相互作用调控胰腺癌的侵袭与转移。因而,深入理解胰腺癌中Integrin及其胞内信号分子之间的这种相互调控作用能够为理解胰腺癌的转移提供新的理论依据。选择性剪接修饰是基因转录后调控的一种重要的分子机制。其异常变化往往会导致肿瘤的发生与发展。因而,解析肿瘤细胞中基因选择性剪接的分子调控机制能够为肿瘤的临床诊治提供潜在的干预靶点。前期研究发现非受体酪氨酸激酶Fyn及Integrinβ1在胰腺癌的转移过程中发挥了十分重要的作用。抑制Fyn的活性能够上调胰腺癌细胞中核内不均一蛋白hnRNP E1以及integrinβ1C剪接体的的表达。由于hn RNP E1具有调控基因选择性剪接的功能,因而我们推测:胰腺癌中Fyn的活性能够通过影响hnRNP E1的表达与功能从而调控integrinβ1基因的选择性剪接,并最终影响胰腺癌的转移。本课题主要研究胰腺癌中hnRNP E1剪接子功能复合物调控integrinβ1基因选择性剪接的分子机制。二、研究方案1.我们首先检测了本研究所收集的76例胰腺癌临床标本中hnRNP E1和integrinβ1剪接体的表达,并将检测结果与胰腺癌病人的临床资料进行了相关性分析;2.我们利用BxPC3和Panc-1胰腺癌细胞分析了抑制Fyn活性对细胞中hnRNP E1和integrinβ1剪接体表达的影响;3.我们通过干预hnRNP E1的表达分析了hnRNP E1对BxPC3和Panc-1胰腺癌细胞中integrinβ1基因选择性剪接的影响;4.我们利用RNA免疫共沉淀、蛋白质免疫共沉淀、质谱分析、RNA干扰实验分析了胰腺癌细胞中hnRNP E1剪接子功能复合物对integrinβ1基因选择性剪接的调控机制;5.我们利用在体与离体的实验方法分析了Fyn通过hnRNP E1调控胰腺癌细胞运动与转移的能力;三、研究结果1.胰腺癌组织中hn RNP E1蛋白的胞浆分布以及integrinβ1A剪接体的表达与胰腺癌的TNM分期、淋巴结转移及肝转移具有显著的相关性并且hn RNP E1蛋白的胞浆分布与integrinβ1A剪接体的表达具有显著的正相关性;2.胰腺癌细胞中Fyn的活性通过PAK1调控hn RNP E1的磷酸化水平及其细胞内分布;并通过调节细胞核内hnRNP E1剪接子功能复合物中hnRNP A1和SF2/ASF的组成比例,调控integrinβ1基因的选择性剪接;3.Fyn通过hnRNP E1调控了胰腺癌细胞的迁移、侵袭与转移能力;四、研究结论胰腺癌中Fyn的活性通过PAK1调控hn RNP E1的磷酸化水平及其细胞内分布;并通过调节细胞核内hn RNP E1剪接子功能复合物中hn RNP A1和SF2/ASF的组成比例,调控integrinβ1基因的选择性剪接,并最终影响胰腺癌的侵袭与转移。
[Abstract]:Background Pancreatic carcinoma is a malignant digestive tract disease with high mortality. Local invasion and distant metastasis of tumor cells occur in the early stage of pancreatic cancer.One of the main clinicopathological features of pancreatic cancer is the presence of a large number of fibrous tissues and abundant extracellular matrix components around the tumor cells.These special ECM components can regulate the invasion and metastasis of pancreatic cancer by activating the interaction of integrin Integrin and its intracellular signaling molecules.Therefore, an in-depth understanding of the interaction between Integrin and its intracellular signaling molecules in pancreatic cancer may provide a new theoretical basis for understanding the metastasis of pancreatic cancer.Selective splicing modification is an important molecular mechanism of gene posttranscriptional regulation.Its abnormal changes often lead to the occurrence and development of tumors.Therefore, the molecular regulation mechanism of gene selective splicing in tumor cells can provide potential intervention targets for the clinical diagnosis and treatment of tumor.Previous studies have found that non-receptor tyrosine kinase Fyn and Integrin 尾 1 play an important role in the metastasis of pancreatic cancer.Inhibition of Fyn activity could up-regulate the expression of hnRNP E1 and integrin 尾 1C splicing proteins in the nucleus of pancreatic cancer cells.Because hn RNP E1 has the function of regulating gene selective splicing, we speculate that the activity of Fyn in pancreatic cancer can regulate the selective splicing of integrin 尾 1 gene by affecting the expression and function of hnRNP E1, and ultimately affect the metastasis of pancreatic cancer.The aim of this study was to investigate the molecular mechanism of hnRNP E1 splicing complex regulating selective splicing of integrin 尾 1 gene in pancreatic cancer.Second, research programme 1.We first detected the expression of hnRNP E1 and integrin 尾 1 splicing bodies in 76 cases of pancreatic cancer, and analyzed the correlation between the results and the clinical data of pancreatic cancer patients.We used BxPC3 and Panc-1 pancreatic cancer cells to analyze the effect of inhibition of Fyn activity on the expression of hnRNP E1 and integrin 尾 1 splicing bodies.We analyzed the effect of hnRNP E1 on the selective splicing of integrin 尾 1 gene in BxPC3 and Panc-1 pancreatic cancer cells by interfering with the expression of hnRNP E1.We used RNA immunoprecipitation, protein immunoprecipitation and mass spectrometry to analyze the regulation mechanism of hnRNP E1 splicing functional complex on integrin 尾 1 gene selective splicing in pancreatic cancer cells.We used in vivo and in vitro experiments to analyze the ability of Fyn to regulate the movement and metastasis of pancreatic cancer cells through hnRNP E1.The cytoplasmic distribution of hn RNP E1 protein, the expression of integrin 尾 1A splice and the TNM stage of pancreatic carcinoma.There was significant correlation between lymph node metastasis and liver metastasis, and there was a significant positive correlation between the cytoplasmic distribution of hn RNP E1 protein and the expression of integrin 尾 1A splice.The activity of Fyn in pancreatic cancer cells regulated the phosphorylation level of hn RNP E1 and its intracellular distribution through PAK1, and the composition of hnRNP A1 and SF2/ASF in hnRNP E1 splicing functional complex.3. Fyn regulates the migration, invasion and metastasis of pancreatic cancer cells through hnRNP E1. 4. Conclusion the activity of Fyn in pancreatic cancer regulates the phosphorylation level of hn RNP E1 and its intracellular distribution through PAK1.The selective splicing of integrin 尾 1 gene was regulated by regulating the composition of hn RNP A1 and SF2/ASF in the functional complex of hn RNP E1 splice, and the invasion and metastasis of pancreatic cancer were also affected.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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