原癌基因CNTN-1/SOX-2介导乳腺肿瘤发生的分子机制

It was a hot and key point to research for effective anti-tumor molecular mechanism of development, invasion, metastasis in recently. CNTN-1 is a kind of neural cell adhesion molecule involved in growth and development, in most cytokines involved in cell differentiation it was potential and a variety of cellular functions mediated by the nervous system. Sox-2 is a transcription factor containing HMGbox and related to SRY protein. Some researchers showed that cell in early embryonic germ expression Sox-2. Sox-2 as a "stem factor" can maintain self-renewal and multi-directional differentiation plays an important role in the progress of embryonic stem cells, combined with the theory of cancer stem cells, which makes it become the hot research field in tumor molecular biology. The current studies had proved that the proto oncogene CNTN-1, Sox-2 and tumor has certain correlation, but its exact mechanism of action is not clear. In this study, with the key CNTN-1 and Sox-2 as a breakthrough point to elaborates the molecular mechanism in the development of mammary gland tumorigenesis, invasion and metastasis as the scientific objectives, to observe the expression of CNTN-1/Sox-2 in canine mammary gland tumor pathological tissue and tumor cell lines, to find the relationship between the gene of CNTN-1/Sox-2 deletion, over-expression and by RNA interference technology explore the gene and tumor cells, tumor stem cell invasion, metastasis, proliferation and tumorigenic. After study for these, we will to understand well from the fundamentally the key role played by CNTN-1/Sox-2 between in the breast primary tumor and metastasis, and further analysis sensitivity of chemotherapy drugs it with tamoxifen, and to provide a theoretical basis for the prevention and control of breast tumor effectively.

CNTN-1为一种神经细胞粘附分子，参与介导神经系统的生长发育及多种细胞功能，是近年来发现最有潜力参与细胞分化的细胞分子。Sox-2是一种与SRY蛋白相关的含有HMGbox的转录因子。Sox-2作为“干性因子”在胚胎干细胞中维持自我更新及在多项分化过程中起决定性作用，结合肿瘤干细胞学说，使其成为在肿瘤分子学领域研究热点，现有的研究证明原癌基因CNTN-1、Sox-2在促进肿瘤发生发展中起一定作用，但其确切作用机制尚不清楚。本研究以关键原癌基因CNTN-1、Sox-2为突破点，阐述其在乳腺肿瘤发生及侵袭转移的关键分子机制，通过观察CNTN1/Sox-2在犬乳腺肿瘤病理组织及相关细胞系中的表达情况，采用基因干扰技术通过使其沉默或过表达，通过体内外实验，探究上述基因与肿瘤细胞、肿瘤干细胞增殖转移和成瘤能力之间的相互关系，为揭示乳腺肿瘤的生成、转移机制及为肿瘤有效预防提供理论依据。

肿瘤的发展进程本质上与原癌基因的激活密切相关。原癌基因广泛存在于生物界的高度保守基因。在生理状态下，处于非激活状态，不具有致癌性。但原癌基因一旦受到体内外致癌因子的作用容易成为促癌基因，这种变化会导致细胞EMT和CSCs特性增强加快肿瘤发展进程。同样，EMT和CSCs特性增强也会导致更多原癌基因被激活，从而导致病程恶性发展。CNTN1和SOX2是重要的原癌基因，我们在犬乳腺癌细胞中沉默CNTN1和SOX2的表达，发现细胞增殖，迁移能力随之下降。进一步验证结果表明主要通过AKT，Hippo及Wnt信号通路抑制肿瘤细胞增殖，迁移和EMT，CSCs特性变化，抑制肿瘤细胞发展。为得到更多可用于诊断和治疗的分子，我们通过免疫组化等方法对犬乳腺癌组织进行收集整理分型，通过RNA测序检测了278个差异表达的miRNAs，而miR-124作为代表因子进行功能验证。结果表明，肿瘤发生过程中涉及多种基因变化，主要通过影响EMT和CSCs特性变化控制肿瘤发展。另外，制备犬her-2-ECD-4，VEGF单克隆抗体，并对上述2种抗体的免疫原性、特异性、稳定性、亲和性等进行了检测，构建了双抗夹心检测方法。此外，我们成功构建了TAM犬乳腺肿瘤耐药细胞，结果表明耐药性的产生也与原癌基因调控EMT和CSCs特性有关。总之，我们探究了原癌基因CNTN1和SOX2的重要作用，筛选了大量肿瘤标记物，验证了miR-124功能作用为以后分子靶向治疗和诊断提供了理论依据。同时，构建了两株单克隆抗体以及TAM耐药细胞系，为日后继续探究犬乳腺肿瘤转移机制及耐药性产生提供了良好的实验基础。