Sox3对食管鳞癌细胞恶性表型的影响及机制研究

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancer worldwide, with little knowledge of its mechanisms and molecular therapeutic targets. We have demonstrated that up-regulation of Sox3 is significantly associated with augmented tumorigenesis and depressed prognosis. Herein, we speculate that Sox3 might up-regulate β-catenin, and then activate Wnt signaling pathway, therefore promote the malignant behavior of ESCC cells. In this study, ESCC cells will be transfected and used as experimental model. Tumor proliferation, apoptosis, invasion and chemoresistance, as well as tumorigenesis and metastasis will be detected and compared in vitro and in vivo in Sox3 positive/negative lentiviral-transduced ESCC cells. Regulation of Sox3 towards β-catenin and the other downstream targets in Wnt signaling pathway will be verified using western blot, RT-PCR, reporter gene assay and co-immunoprecipitation, action sites of Sox3 by which β-catenin regulated will be localized using site-directed mutagenesis technique, and the mechanism will be verified by blocking and interference of β-catenin. At last, curative effect of Sox3-targeted therapy will be evaluated and compared with traditional chemotherapeutic drugs. This study might be helpful in revealing the mechanism of Sox3/β-catenin in ESCC, and exploring its novel therapeutic targets.

食管鳞癌（ESCC）是严重威胁人类健康最常见的恶性肿瘤之一，其机制和靶向药物尚不明确。我们已证实Sox3表达增高与ESCC的发生、发展和预后密切相关。我们假设：Sox3可通过上调β-catenin激活Wnt经典信号通路，从而增强ESCC细胞的恶性表型。本研究拟以ESCC细胞系为研究模型，通过体内外实验，检测Sox3表达改变对ESCC细胞增殖、凋亡、侵袭、化疗耐药以及肿瘤形成和转移的影响；通过Western blot、RT-PCR、报告基因、免疫共沉淀等明确Sox3对β-catenin及其下游信号分子的调控，并通过定点突变确定Sox3调控β-catenin的作用位点，再通过β-catenin阻断剂和siRNA干预对上述机制进行检验；最后通过与化疗药物对比，评价Sox3靶向治疗在ESCC中的效果。本研究有助于揭示Sox3/β-catenin在ESCC中的作用机制，探索ESCC新的治疗靶点。

食管鳞癌(ESCC)是严重威胁人类健康最常见的恶性肿瘤之一，其机制尚不清楚。本研究以ESCC组织、细胞和动物为研究对象，通过体内外实验，初步探讨了SOX3表达改变对ESCC细胞生物学行为的影响，及其在ESCC发生、发展和转移过程中的作用。..一、通过Western blot、MTT、划痕愈合、Transwell、流式细胞术凋亡实验发现：1. SOX3表达降低对ESCC细胞增殖、迁移和侵袭能力起抑制作用，而对ESCC细胞凋亡起促进作用。2. SOX3抑制后Wnt/β-catenin信号通路关键因子p-GSK3βser9、β-catenin、TCF4、c-myc和cyclin D1蛋白表达降低。SOX3可通过Wnt/β-catenin信号通路促进ESCC细胞的增殖、迁移和侵袭。.二、通过裸鼠移植瘤实验、Western blot、ELISA、免疫组化、淋巴管内皮细胞成管实验发现：1. Sox3可促进ESCC细胞的淋巴管内皮细胞成管能力。2. SOX3对ESCC细胞体内成瘤作用不明显，但可通过VEGF-C和VEGF-D促进ESCC淋巴结转移。3. SOX3可通过VEGF-C/VEGF-D/VEGFR-3通路促进ESCC淋巴管的生成。.三、通过MTT细胞增殖实验、划痕愈合实验、鸡胚绒毛尿囊膜实验、Western blot、ELISA、萤光素酶报告基因实验和裸鼠移植瘤实验发现：1. CCR7可促进ESCC细胞的增殖和血管内皮细胞的迁移，增强培养上清液诱导血管形成能力，并上调VEGF-A和VEGF-C蛋白的表达。2. CCR7可增强NF-κB报告基因的转录活性，并促进p-IKK、p-IkBα、TNF-α、IL-6、IL-8和TGF-β表达。3. CCR7可通过NF-κB信号通路促进ESCC移植瘤生长增殖。.以上结果表明，SOX3和CCR7对ESCC的恶性表型起促进作用，并可分别通过Wnt/β-catenin、VEGF-C/VEGF-D/VEGFR-3和NF-κB信号通路促进ESCC的增殖、迁移和侵袭，以及肿瘤淋巴管和血管形成。本研究对阐释ESCC发生、发展和转移的机制具有一定价值。