DENND2D的诱导表达对非小细胞肺癌细胞恶性表型影响及其作用机制研究

DENND2D（DENN/MADD domain containing 2D）was identified as being down-regulated in lung cancer low-expression suppression subtractive hybridization library, we first report the relationship between this gene and lung carcinogenesis, but its phenotype and mechanism is still unclear. Now we have constructed the Tet-On inducible expression adenoviral system of DENND2D, and pre-experiment data shows that induced expression of DENND2D can directly induce cell apoptosis, and the apoptosis incidence presented in manner of the time-dose dependent of DENND2D expression level; and DENND2D increase the activity of MAPK-specific luciferase reporter，moreover，DENND2D increase the key kinase JNK phosphorylation level. So the hypnosis is that whether the DENND2D induced apoptosis is regulated through MAPK pathway. Here is our research plan: firstly we will observe the change of cell phenotype after DENND2D induced expression including anchorage-independent growth, invasion and metastasis, nude mouse transplantation tumorigenesis et al; then we will study on the relationship between DENND2D induced expression and cell apoptosis including the change of mitochondria membrane potential, and the activity of Caspase et al; we will also study the relationship between DENND2D induced expression and the activation of MAPK pathway; lastly we will inject the inducible adenovirus to nude mouse transplantation tumor, estimate the application potential of DENND2D in biotherapy, meanwhile we will detect the apoptosis incidence and MAPK pathway activation in transplantation tumor samples, to validate the scientific hypothesis in vivo.

DENND2D（DENN/MADD domain containing 2D）源自非小细胞肺癌抑制性消减杂交低表达文库，我们在国际上首次报道该基因与肺癌的相关性，并成功构建DENND2D的四环素诱导表达腺病毒系统，预实验DENND2D可以诱导细胞凋亡，同时DENND2D增加MAPK通路特异的报告基因活性，并增加通路关键激酶JNK磷酸化水平。但该基因表型及机制目前仍属未知，尤其DENND2D的诱导凋亡机制以及是否由MAPK信号通路介导是我们关心的科学问题，为此拟开展如下研究：首先观察DENND2D对肿瘤细胞恶性表型影响；进一步分析DENND2D诱导表达与细胞凋亡的关系，包括线粒体膜电位变化、凋亡通路中关键分子的活化情况等；阐明DENND2D活化MAPK信号通路的主要途径，深入检验DENND2D诱导凋亡与MAPK通路活化的关系。最后，利用裸鼠移植瘤模型在in vivo水平验证我们的科学假设。

DENND2D（DENN/MADD domain containing 2D）源自非小细胞肺癌抑制性消减杂交低表 达文库，为探讨其基因表型及机制我们进行了系列工作。首先我们利用Westernblot和免疫组化的方法，在临床样本中检测到了DENNDD蛋白水平的表达降低。进一步我们成功构建了DENND2D的四环素诱导表达腺病毒系统，细胞生物学实验显示DENND2D诱导表达后可以抑制肺癌细胞系的生长及锚定非依赖生长，裸鼠移植瘤实验从体内水平证实了DENND2D的抑瘤作用。为研究其作用机制，我们利用流式细胞术检测了PS外翻、线粒体膜电位等，发现DENND2D可以直接诱导细胞凋亡并且存在量效关系，Westernblot检测到了凋亡通路中的关键激酶Caspase9和Caspase3的活化。应用广谱Caspase抑制剂和针对Caspase 9的抑制剂后，DENND2D的凋亡诱导作用明显下调，初步证实DENND2D可以直接诱导Caspase依赖的线粒体途径凋亡。为探索DENND2D的细胞信号通路作用机制，我们开展了系列研究，结果显示DENND2D诱导表达增加了MAPK通路特异的报告基因活性，并增加通路关键激酶JNK磷酸化水平。应用JNK显性负突变体和特异JNK抑制剂SP600125均未对DENND2D的凋亡诱导作用产生影响，提示DENND2D的凋亡诱导作用可能并非JNK依赖。最后我们利用Tunel方法原位检测到裸鼠移植瘤内凋亡的增加，体内水平回答了DENND2D肿瘤抑制的可能机制。利用上述细胞生物学手段，我们初步回答了DENND2D的抑癌属性和作用机制，希望可以为肺癌临床诊疗提供新的靶标。