缺氧微环境下PAK1对大肠癌EMT效应影响的基础研究

PAK1 is a kind of evolutionarily conserved serine/threonine protein kinase, its kinase activity and subcellular localization is closely related to function, with high expression in a variety of tumors. The relationship between PAK1 and EMT is unknown. HIF-1 alpha plays a key role in tumor hypoxia microenvironment and is correlated with EMT. Preliminary work was first found in hypoxia microenvironment PAK1 was upregulation in colorectal cancer cells, interfering PAK1 can influence EMT actuating markers E-cadherin and key transcription factor Snail expression. This prompted in hypoxic microenvironment PAK1 may participate in EMT of colorectal cancer. This project aims to combine the co-transfection, immunofluorescence and dual luciferase report gene technology, orders colorectal cancer cell lines and clinical colorectal cancer tissue specimens as the research object, research on the correlation between PAK1 and HIF-1 alpha,EMT markers in colorectal cancer; the relationship between PAK1's subcellular localization,kinase activity and EMT in hypoxia microenvironment;PAK1 is synergistically promote hypoxia ( HIF-1 alpha ) induced EMT or not; the relationship between PAK1 and Snail's promoter activity, Snail's cellular localization and its key regulatory molecules GSK-3 beta, NF kappa B. This study maybe provide new theoretical basis for colorectal cancer' biotherapy.

PAK1为一类进化上保守的丝氨酸/苏氨酸蛋白激酶，其激酶活性及亚细胞定位与功能密切相关，在多种肿瘤中表达增高。PAK1与肿瘤EMT关系不明。HIF-1α在肿瘤缺氧微环境中起着中枢纽带作用且与EMT密切相关。前期工作首次发现缺氧可诱导大肠癌细胞中PAK1表达上调，干扰PAK1可影响EMT始动标志E-cadherin及关键转录因子Snail的表达。这提示缺氧微环境下PAK1可能参与大肠癌EMT。故本项目拟结合共转染、免疫荧光及双荧光素酶报告基因等技术，以大肠癌细胞株及临床大肠癌组织标本为研究对象，研究①PAK1与HIF-1α、EMT标志分子在大肠癌组织中的相关性；②缺氧时PAK1亚细胞定位及激酶活性与EMT的关系；③PAK1是否协同促进缺氧(HIF-1α)诱导的EMT；④PAK1对Snail启动子活性、细胞定位及其关键调控分子GSK-3β、NF-κB之间的关系。可为大肠癌生物治疗提供新理论依据

研究表明PAKs生物学功能的发挥受到Rac1、PI3K/AKT及EGF等多种分子调控，且与其亚细胞定位存在密切关系。HIF-1α在缺氧微环境中起着中枢纽带作用。前期工作证明：缺氧时大肠癌细胞中PAK1,5表达增高；干扰PAK1,5表达可影响EMT关键蛋白及转录因子表达，这些提示缺氧微环境下PAK1,5参与大肠癌的EMT效应。缺氧时PAKs是否依赖于HIF-1α来调控EMT及具体机制不明，有待研究。本项目通过免疫组化检测证实了PAK1，HIF1α表达与大肠癌的侵袭转移潜能密切相关。大肠癌组织中PAK1、HIF1α表达分别与Snail， Ecad之间存在显著的相关性。PAK1与HIF1α之间的表达显著相关。2、氯化钴诱导下成功构建了缺氧微环境下大肠癌细胞发生EMT的模型。3、缺氧微环境下PAK1可以调控Snail介导大肠癌细胞发生EMT。4、缺氧微环境下大肠癌细胞中，PAK1与HIF1α之间存在相互调控关系，二者共同参与到对关键转录因子Snail表达的调控，而介导EMT。5、缺氧微环境下大肠癌细胞中， PAK1 可通过调控NF-κB，继而调控关键转录因子Snail使细胞发生EMT效应。综上所述，本研究的科学意义在于探讨缺氧时PAK1调控大肠癌细胞发生EMT的机理，可为大肠癌发生浸润、转移提供新的理论和实践依据，有利于大肠癌的早期诊治及改善预后。.

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