CD147调控Ca2+/NFAT1信号通路促进恶性黑色素瘤侵袭转移的机制研究

Our previous experiments found that calcium-modulating cyclophilin ligand（CAML）interacted with CD147 in the endoplasmic reticulum（ER）of human malignant melanoma cells using the yeast two-hybrid system. And CD147-CAML complex can increase the intracellular Ca2+ levels by promoting Ca2+ efflux from ER to cytoplasm, then activate the related signals and effectors. And then the upregulation of MMP9 can be induced. At the same time, the activity of NFAT1, a Ca2+-dependent transcript factor, was downregulated by knockdown of CD147 expression. And knockdown of NFAT1 by siRNA can also decrease the expression of MMP9. Therefore, a series of expriments will be conducted to identify the relationships between CD147, CAML and plasma membrane Ca2+ channels, and then elucidate the mechanisms of CD147 in upregulating the cytoplasmic Ca2+ concentration. Subsequently, the activities of NFAT1 under different conditions will be analyzed to identify the way of CD147 in activating the NFAT1. Later, The roles of NFAT1 in maintaining characteristics of melanoma cells and its relationship with the CD147-mediated MMP9 expression will be studied by Chip and Luciferase gene reporter system in control melanoma cells and NFAT1 knockdown cells. Finally, the possibility of together CD147 with NFAT1 and CAML as markers for early diagnosis and prognosis of melanoma will be studied by statistical analysis of clinical documents and IHC staining results. The expected results not only are helpful to elucidate the roles and molecular mechanisms of CD147/Ca2+/NFAT1 pathway in the metastasis of malignant melanoma, but also can provide candidate molecular targets for targeted therapy of human malignant melanoma. It has important theoretical significance and practical application value.

我们前期的研究首次发现，黑色素瘤细胞中CAML是CD147的相互作用蛋白， CD147通过结合CAML能上调胞浆Ca2+的浓度和MMP-9的表达，预实验用转录因子芯片发现敲降CD147能降低NFAT1的活性。因此，我们假设 "CD147通过与CAML相互作用，增加胞质内Ca2+浓度，激活CaN-NFAT1，诱导MMPs的表达，促进黑色素瘤的侵袭转移"。为此，本项目拟分析CAML在CD147上调胞质内Ca2+浓度以及Ca2+在CD147活化CaN-NFAT1中的作用；分析NFAT1在CD147诱导MMPs表达中的作用；分析CD147、CAML、CaN、NFAT1和MMPs在黑色素瘤临床样本中的表达及临床意义。本项目研究不仅将助于揭示CD147通过与CAML相互作用介导Ca2+/NFAT1/MMPs信号通路在黑色素瘤侵袭转移中的作用及分子机制，而且能为黑色素瘤的侵袭转移预警和靶向治疗提供靶标。

恶性黑素瘤是由皮肤和其他器官黑素瘤细胞产生的一种发病率不断提高而预后却较差的恶性实体肿瘤。黑素瘤发病隐匿，多数患者就诊时已发生肿瘤转移，癌细胞的侵袭转移已成为影响黑素瘤患者预后的关键因素。CD147是一种免疫球蛋白超家族成员的新型跨膜糖蛋白，在包括恶性黑素瘤在内的多种肿瘤组织中高表达，与肿瘤侵袭转移密切相关。根据文献研究表明CD147可以通过调节Ca+信号进而促进肝癌侵袭转移，而在黑素瘤中的作用仍未阐明。因此我们展开了CD147与CA/NFAT1信号对黑素瘤侵袭转移的研究。主要通过体外在黑素瘤细胞中干扰CD147及NFAT1的表达，检测二者之间的关系及对黑素瘤侵袭转移的影响，同时体内转移瘤实验验证NFAT1对黑素瘤侵袭转移的影响。重要结果数据如下：1、首先我们明确了CD147可活化钙依赖性磷酸酶-NFAT1信号；2、稳定敲低CD147后可抑制CAN酶的活性（酶活性检测）；3、稳定敲低CD147后提取核蛋白WB检测、免疫荧光结果均表明CD147可抑制NFAT1活化入核；4、稳定敲低NFAT1后可显著抑制MMP的mRNA及蛋白表达，减弱黑素瘤细胞的侵袭转移能力（与动物转移瘤模型结果一致）；5、其后的RESCUE实验恢复NFAT1表达后可部分恢复黑素瘤细胞的侵袭转移能力；6、荧光素酶报告基因实验及CHIP实验结果表明NFAT1可直接与MMP9结合；7、最后在黑素瘤患者与正常人组织芯片结果表明CD147与NFAT1在黑素瘤病人组织中相对高表达，且二者具有显著的相关性，二者呈正相关。通过以上研究证实了CD147可通过CAN钙离子信号通路调控NFAT1活化，进而影响黑素瘤侵袭转移，有助于揭示CD147上调Ca/NFAT1信号在黑素瘤侵袭转移中的作用及其分子机制，并为黑素瘤的侵袭转移预警和靶向治疗提供靶标。