NKX2.1调控MKK4触发EMT在结直肠癌侵袭转移中的作用机制研究

The MKK4 gene involved in tumor development, but its role in tumor invasion and metastasis mechanism remains unclear. Our previous study found that MKK4-1304T> G single nucleotide polymorphism associated with cancer susceptibility, and then found that associated with colorectal cancer invasion and metastasis. Further, bioinformatics analysis and function experiments revealed that the transcription factor NKX2.1 may regulate the expression of MKK4 on the basis of MKK4 polymorphisms, while the expression of MKK4 changes affect epithelial mesenchymal transition (EMT) markers. Therefore, based on the previous research results, we proposed the scientific hypothesis that "NKX2.1 based on MKK4-1304T>G locus polymorphisms regulated the MKK4, and then activate EMT pathway to promote or inhibit the invasion and metastasis of colorectal cancer". In order to verify this hypothesis , The project intends to carry out the following research: first, to explore the correlation of MKK4-1304T> G polymorphism, MKK4, NKX2.1 expression levels and colorectal cancer invasion and metastasis; Second, to clarify the molecular mechanisms of NKX2.1 regulated the expression of MKK4 on based of MKK4-1304T> G polymorphisms state; Finally, to explore the mechanism that NKX2.1/MKK4 via EMT pathway to promote or inhibit colorectal cancer invasion and metastasis. This study will help to reveal the molecular mechanism of NKX2.1/MKK4 pathway in colorectal cancer invasion and metastasis, its completion will provide new candidate markers for CRC diagnosis and treatment, and new potential drug targets for treatment of CRC.

MKK4基因参与肿瘤发生发展，但其在肿瘤侵袭转移中的作用机制仍不明确。我们前期发现MKK4-1304T>G位点与肿瘤易感性相关，进而发现其与肠癌侵袭转移相关，生物信息学分析及功能实验揭示转录因子NKX2.1可能基于多态性位点调控MKK4，而MKK4表达改变影响上皮间质转化（EMT）相关标志物。因此，我们提出"NKX2.1基于MKK4-1304T>G位点多态性调控MKK4触发EMT途径促进或抑制肠癌的侵袭转移"的科学假设。并围绕该假设开展以下研究：探究MKK4-1304T>G位点多态性、MKK4、NKX2.1表达水平与肠癌侵袭转移的相关性；阐明NKX2.1基于MKK4-1304T>G位点多态性调控MKK4表达的分子机制；探讨NKX2.1/MKK4通过EMT途径促进或抑制肠癌侵袭转移机制。本研究将有助于揭示 NKX2.1/MKK4通路在肠癌侵袭转移中的分子机制，为指导肠癌靶向治疗提供科学依据。

MKK4基因参与肿瘤发生发展，但其在肿瘤侵袭转移中的作用机制仍不明确，我们前期发现MKK4-1304T>G位点与肿瘤易感性相关，并通过生物学分析手段推测NKX2.1可能基于多态性位点调控MKK4，而MKK4表达改变影响上皮间质转化（EMT）相关标志物的表达。本研究围绕上述问题展开了相关研究，首先分析前期已建立的临床病例队列，结合病例资料和随访资料综合评价MKK4-1304T>G多态性位点与结直肠癌侵袭转移之间的临床相关性，发现MKK4 -1304T基因型与结直肠癌侵袭转移相关，该基因型携带者总体生存时间明显短于MKK4 -1304G基因型携带者。. 前期研究发现MKK4-1304T>G会影响MKK4的表达，然而MKK4基因在肿瘤发生发展中以及肿瘤侵袭转移中的作用机制不明确，我们首先通过TCGA、GEO等数据库分析MKK4的表达谱与结直肠癌的发生发展以及预后的关系，并在21对结直肠癌组织及癌旁组织qpcr、wb进行后续验证，研究发现MKK4在肿瘤组织中表达低于癌旁组织（P<0.05）,但在腺瘤组织与瘤旁组织表达并无明显差异（P>0.05）。生存分析发现MKK4高表达与患者预后良好相关，但并无统计学差异。我们继而构建肠癌组织的试验组和验证组组织芯片，发现pMKK4表达与临床病理因素及患者预后密切相关，研究成果已整理发表在《Oncotarget》。据此，我们推测MKK4在结直肠癌中扮演着抑癌基因的角色。为了进一步证明MKK4的抑癌功能，我们构建了MKK4稳定表达的细胞株（DLD1、HCT8），并开展了一系列的细胞功能试验，发现MKK4与肿瘤增殖、侵袭转移以及5-FU耐药密切相关。因此，我们推测MKK4在肿瘤的发生发展过程中，受到了更为复杂的调控，有待更进一步的研究。

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