EphA3和EphB6在大肠上皮细胞恶性转化和演进中的作用及其调控机制

As one of the largest class of tyrosine receptor family, Eph is a potential drug target for treatment, but the specific role and its molecular mechanisms of its family members in tumor are poorly understood.Our preliminary study found that abnormal expression of EphA3 or EphB6 can influence the biology behaviors of colorectal epithelial cell, including cell proliferation, invasion and metastasis. It suggestes that EphA3 and EphB6 may be involved in malignant transformation and evolution of colorectal cells. On the basis of early work, by using site-directed mutagenesis technology, the applicants will construct a series of relatively common mutant recombinants of EphA3 and EphB6. Four colorectal epithelial cell models from transgenic rat on behalf of colorectal cancer in different pathological processes and different context of APC and Ras gene expression,are used as the experimental object. The role of EphA3 and EphB6 on malignant transformation and evolution of colorectal epithelial cell will be studied by using microarray,proteomics and bioinformatics. Further study will be carried out to clarify its signal transduction pathways and molecular mechanism. The above study will provide the theoretical basis and new ideas for the prevention, diagnosis and treatment for colorectal cancer.

Eph是酪氨酸受体家族中的一大类，是潜在的药物治疗靶标，但其家族成员在肿瘤中发挥的具体作用及分子机制却知之甚少。申请人前期发现该家族成员EphA3、EphB6突变或异常表达可影响大肠上皮细胞生物学行为，在细胞恶性增殖、侵袭和转移中发挥重要作用，提示EphA3和EphB6可能参与了大肠细胞恶性转化和演进过程。本项目拟在已有工作基础上，采用定点突变技术构建EphA3和EphB6常见突变体，以4种分别代表大肠癌不同病理进程的含有APC或/和RAS突变的转基因鼠模式细胞为材料，采用基因芯片与蛋白质组学，结合生物信息学技术，研究EphA3和EphB6突变或异常表达对大肠上皮细胞恶性转化及演进的影响，探讨其在大肠癌发生发展过程中的作用机制，阐明其影响的信号转导通路，为大肠癌的预防、诊断和治疗提供理论基础和新思路。

Eph是酪氨酸受体家族中的一大类，是潜在的药物治疗靶标。本课题组发现该家族成员野生型EphB6在大肠腺瘤细胞中过表达，结合APC基因突变可促进细胞恶性增殖、侵袭和转移。采用基因芯片结合生物信息学技术，提示恶性转化有关的差异基因表达主要与P53和Pathway in cancer信号转导途径有关。野生型EphA3及其突变体EphA3-T37K在腺瘤细胞中过表达时，细胞表面超微结构发生了明显变化，促进细胞侵袭和迁移，并获得裸鼠皮下成瘤能力，提示EphA3异常表达促进大肠腺瘤细胞恶性转化。利用高通路芯片筛选转染前后IMCE细胞差异表达mRNA和lncRNA，经过Go(Gene Ontology)分析和KEGG Pathway分析，与恶性转化有关的差异基因表达主要与MAPK和VEGF信号转导途径有关。挑选差异表达显著的mRNA和lncRNA绘制lncRNA-mRNA共表达网络，筛选关键基因(hub genes),排名前5位的关键基因按顺序分别是GRB2、BRCA1、NFKB1、SFN和FN1。文献报道GRB2、NFKB1、SFN和FN1均与细胞外环境有密切关系，故进一步研究EphA3对肿瘤微环境的影响及相关机制，将为大肠癌的预防、诊断和多靶点治疗提供理论基础和新思路。我们的研究结果揭示了在结直肠的细胞中的EphB6及EphA3基因的致瘤性活动机制，可以为结直肠癌提供新奇的预防目标，早期诊断以及治疗措施。