摘要
NECL1(Nectin-like molecule 1),又名CADM3/IGSF4B/TSLL1/SynCAM3,是神经系统特异表达的免疫球蛋白样的细胞粘附分子。它是一个单跨膜蛋白,其中包含N端3个Ig-like结构域组成的胞外区、单一跨膜区及短的C末端胞内区,其N端可以同自身或同家族成员形成同源或异源二聚体,C末端可以同胞内4.1蛋白家族成员和包含PDZ结构域的蛋白发生相互作用。目前的研究报道提示,NECL1在突触形成、轴突髓鞘形成、小脑形态发生中发挥重要的作用。
曾经有人报道NECL1在13例人神经胶质细胞瘤细胞系中表达缺失,提示NECL1在神经胶质瘤发生中可能发挥一定的功能。在此,我们利用荧光实时定量RT-PCR和免疫组织化学的方法检测了在神经胶质瘤细胞系和原发性肿瘤组织中NECL1的表达变化,结果显示NECL1在胶质瘤细胞系和原发性肿瘤组织中确实发生缺失。为进一步研究NECL1可能的功能,我们构建了NECL1的重组腺病毒,利用病毒感染神经胶质母细胞瘤的效率可以达到90%。此后,我们利用流式细胞术和BrdU掺入的方法发现在U251中恢复表达NECL1后可以抑制细胞周期的进展并造成DNA合成的阻滞。裸鼠体内成瘤实验同样证明恢复表达NECL1可以抑制U251细胞的成瘤能力,限制肿瘤生长。我们还观察到U251细胞表达NECL1后在高密度培养情况下其细胞形态可以发生变化,于是又检测了NECL1和肿瘤细胞迁移和侵袭的关系。利用划痕伤害实验(Wound line)和Transwell实验,我们发现NECL1在U251细胞中的恢复表达可以在体外条件下抑制细胞的迁移和侵袭能力,明胶酶谱实验(GelatinZymography)也发现MMP2和MMP9蛋白的活性同样发生了变化。因此,我们初步推论NECL1可能作为一个胶质瘤发生的抑制因子。
在上述工作基础上,我们对NECL1抑制神经胶质瘤增殖、迁移的分子机制进行了初步探讨。我们选择DNA基因芯片技术筛选在NECL1恢复表达的情况下U251细胞内基因表达谱的变化。一共筛选到204个表达差异基因,其中184个表达下调,20个表达上调。根据生物信息学预测和相关文献报道,我们发现若干细胞外基质蛋白的表达发生了变化,如OPN,CCL2等。以OPN作为代表,我们对其表达情况及下游作用分子进行了验证,结果和芯片提示相符。接下来,我们利用NECL1恢复表达的U251细胞条件化的培养基处理细胞,利用上述的相关实验方法进行验证发现条件培养基可以部分解除NECL1对U251细胞的抑制作用.提示细胞外基质中确实含有影响NECL1功能的因子。
除胞外分子作用外,我们同样尝试寻找NECL1的直接下游信号通路变化。我们采用了酵母双杂交技术筛选NECL1下游直接相互作用蛋白。一共得到了9个阳性克隆,包括5个已知序列的蛋白。遗憾的是,在考虑蛋白表达时空的前提下,我们认为这些候选基因与NECL1在胶质瘤中的功能无直接关系。根据文献报道,初步实验发现NECL1的恢复表达可以引起U251细胞内Cdc42活性状态的变化,提示Cdc42可能是NECL1胞内的效应分子之一。更深入的机制需要进一步实验证据的支持。
NECL1(Nectin-like molecule 1),also named as CADM3/IGSF4B/TSLL1/ SynCAM3,is a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule.It is a single transmembrane protein,including three Ig-like domains as extracellular domain located at N terminus,one transmembrane motif and a short C terminal tail.Extracellular domain of NECL1 could form homo-or heterodimer with itself and other members in the family.C terminal tail could interact with both 4.1 protein family members and protein containing PDZ domain.Until now,it has been reported that NECL1 could play some role in synaptogenesis,myelination and cerebellum morphorlogenesis.
Others had reported that NECL1 was abrogated from 13 human glioma cell lines comparing with normal tissue,which suggests that NECL1 may function in glioma generation.In our report,we applied Real time RT-PCR and immunohistochemistry to detect the expression of NECL1 in both glioma cell lines and primary glioma tissues.The result indicated that NECL1 was indeed lost in glioma cell lines and primary glioma tissues. For further study of the function of NECL1,we constructed recombinant adenovirus to induce NECL1 into glioma cell lines.The efficiency of infection was over 90%.Then flow cytometry and BrdU incorporation assay were employed to find that restoration of NECL1 in U251 would inhibit cell cycle process and DNA synthesis.It was also suggested that NECL1 could suppress tumor growth of U251 in nude mice.Additionally,the morphology of cells was changed when the U251 cells were cultured at high density with NECL1 re-expression.Wound line and Transwell assays were performed to study the function of NECL1 in U251 migration and invasion.The results indicated that NECL1 would inhibit cell migration and invasion in vitro.The result of Gelatin zymography assay suggested that restoration of NECL1 would influence the activity of MMP2 and MMP9.Therefore,we concluded that NECL1 may work as a potential tumor repressor in glioma generation.
For studying the mechanism of NECL1 inhibiting cell proliferation and migration in glioma cell line,DNA gene biochip was applied to screen the different gene expression pattern of U251 with restoration of NECL1.204 differently expressed genes were found, including 184 down-regulated and 20 up-regulated.According to bioinformatic prediction and references report,we found the expression of several extracellular matrix proteins was changed,such as OPN,CCL2 and so on.Selecting OPN as the most important candidate, we examined the expression of OPN and its downstream molecules.The result was as expected.Then the conditional medium of U251 with NECL1 re-expression was collected and employed to treat cells.It was found that conditional medium could partly rescue the phenotype of U251 with NECL1 comparing with the wild type cells,which suggested some important factors influencing the function of NECL1 may exist in extracellular matrix.
Besides extracellular molecules,we also try to look for the direct downstream signaling pathway proteins of NECL1.Yeast two-hybrid assay was performed to screen downstream interacting proteins of NECL1.Nine positive clones were obtained,including five existed proteins.Unfortunately,according to references,we did not think that there was any direction relationship between NECL1 with those candidate genes considering the spatio-temperal expression pattern of genes.Additionally,our preliminary data indicated that restoration of NECL1 in U251 would promote the active form of Cdc42 to shift to the inactive form,which suggested Cdc42 might work as one of downstream factors of NECL1 in U251.More data should be provided for further mechanism.
引文
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