DEC1通过调控claudin家族成员转录促进乳腺癌侵袭转移的分子机制

We reported that the overexpression of Differentiated embryo-chondrocyte-expressed gene 1 (DEC1) was correlated with the malignant phenotype in breast cancer. In our preliminary study, we found DEC1 was overexpressed in the Triple-negative breast cancer compared with other types of breast cancer. In addition, DEC1 knockdown by siRNA could enhance the transcription of claudin-1，-4，-7 and in turn reverse the EMT phenotype induced by TGF-β and attenuate the invasion and metastasis of breast cancer. We analyzed the promoter region of claudins and found E-box motifs which can be recognized by DEC1. We hypothesize that DEC1 inhibits the transcription of claudins through binding to the E-box motif in the promoter region of claudins. To test our hypothesis，we will bidirectionally regulate the transcription of DEC1 and also overexpress E-box-binding deficient mutants in multiple breast cancer cell lines. By adopting ChIP, luciferase reporter assay, PCR, IB, Transwell and nude mouse model, we will try to clarify the detailed mechanism by which DEC1 regulates claudins and enhances the invasion and metastasis in breast cancer. Based on these studies, we hope to find potential targets for molecular therapy and drug design, and further provide theoretical and experimental basis for individual treatment to breast cancer.

我们报道了DEC1表达上调与乳腺癌恶性表型相关, 并发现其在三阴性乳腺癌中表达明显高于其它分子亚型。干扰内源性DEC1可从mRNA和蛋白水平上调claudin-1，-4，-7并阻止由TGF-β诱导的EMT表型，进而抑制乳腺癌细胞的侵袭能力。但DEC1对于claudins的具体调控机制不清。通过序列分析对比，我们推测DEC1通过其BHLH功能域识别和结合claudins启动子区E-box元件以抑制其转录，从而促进EMT并上调乳腺癌的侵袭转移。我们拟通过双向调控乳腺癌细胞系中DEC1的表达，分别构建并转染DEC1和claudins的突变体质粒，应用ChIP、荧光素酶报告基因、PCR、免疫印迹、Transwell及裸鼠移植等方法，阐明DEC1下调claudins转录的分子机制及对乳腺癌细胞侵袭转移的影响,为寻找乳腺癌新的治疗靶点提供依据。

背景：女性乳腺癌发病率的升高与高强度工作所带来的生物节律紊乱有着紧密的联系，而作为生物节律调控基因家族的重要成员DEC1和DEC2在乳腺癌发病中的重要作用逐渐被人们认识。.主要研究内容：观察DEC1和DEC2在乳腺癌各分子亚型中的表达情况。双向调控DEC1和DEC2的表达，观察乳腺癌细胞侵袭转移能力的改变。通过构建DEC1和DEC2的不同剪切体，找出DEC1和DEC2调控EMT的关键结构域。.重要结果：DEC1是乳腺癌侵袭转移的促进因子，而DEC2属于抑制因子。与其它分子表型乳腺癌相比，DEC1高表达同时DEC2低表达的免疫表型在三阴性乳腺癌中的比例较高，并且与肿瘤的高分期，淋巴结转移和不良预后相关。DEC1和DEC2对于EMT相关因子及肿瘤侵袭转移的调控主要是通过BHLH结构域实现的。而DEC1和DEC2在肿瘤中的差异表达，受肿瘤自分泌和旁分泌途经通过TGF-beta信号通路介导从而实现。.关键数据及科学意义：对于乳腺癌非根治性标本，可以通过免疫组化观察DEC1/DEC2的比值，可以帮助预测肿瘤的不良生物学行为，合理采取术后辅助治疗。DEC1与DEC2可能成为三阴性乳腺癌的潜在治疗靶点。