结肠癌中CacyBP/SIP入核后调控蛋白的定量蛋白质组学筛选及鉴定

The mechanism of colon cancer is still unclear. CacyBP/SIP is a newly discovered protein which could interact with S100 proteins family via Ca2+-dependent function,and it's nuclear translocation and phosphorylation depend on the Ca2+ concentration.In the previous study we found:CacyBP/SIP highly express in colon carcinoma tissues and stain in the nuclear; CacyBP/SIP could translocate into nuclear in the colon cancer cell via exogenous stimulation; While CacyBP/SIP nuclear translocation could promote the proliferation of colon cancer cell. Tandem Affinity Purification screening downstream signaling molecules don't obtain positive result. Structural analysis suggests that: CacyBP/SIP interacts with Skp1, S100, ERK1/2 and the other two target proteins. So this project intends to use SILAC (stable isotope labeling by amino acids) to screen the protein complex interacting with CacyBP/SIP after its nuclear translocation combined with mass spectrometry; and also screen the up-regulated or down-regulated target gene using related signal pathway gene chip.Through the experiment in vitro pull-down and in vivo co-IP, build the network diagram between CacyBP/SIP and its interacted proteins,and then reveal the molecular mechanisms of CacyBP / SIP after nuclear translocation.Then we could provide experimental basis for the treatment of colon cancer on a molecular level and inhibit cancer cell proliferation at the source.

结肠癌发生机制不清。钙周期素结合蛋白（CacyBP/SIP）是新发现的以钙依赖方式作用的S100家族的靶蛋白，具有依赖Ca2＋浓度的核转位及磷酸化。我们前期发现：CacyBP/SIP在结肠癌组织中高表达，胞核着色；且在结肠癌细胞中具有核转位，并导致癌细胞增殖；TAP法筛选下游信号分子，未获得阳性结果。结构分析提示：CacyBP/SIP可以与Skp1、S100和ERK1/2等五个靶蛋白结合。本课题拟通过大规模、高通量的蛋白质相互作用技术SILAC法，结合质谱分析，筛选出CacyBP/SIP入核后结合的蛋白复合体；通过相关信号通路基因芯片筛选CacyBP/SIP入核后上调/下调靶基因；经体内外蛋白结合实验来鉴定，从而构建CacyBP/SIP入核后相互作用蛋白的网络图，以图揭示CacyBP/SIP入核后促结肠癌发生发展的分子机制，为在源头上抑制结肠癌增殖，达到分子水平上治疗结肠癌提供实验依据。

目的:钙周期素结合蛋白（Calcyclin-binding protein， CacyBP/SIP）主要表达于细胞质中，本课题组前期研究发现，胃泌素可刺激结肠癌细胞SW480中CacyBP/SIP发生核转位，且促进细胞增殖。本研究为进一步筛选CacyBP/SIP入核后下游相互作用的靶蛋白，探索CacyBP/SIP入核后下游结合分子的信号通路，探究其在结肠癌发生发展中的可能作用。.方法:构建CacyBP/SIP核转位细胞模型，用激光共聚焦扫描显微镜术和Western Blot验证细胞模型成功建立。提取造模组与未刺激组核蛋白，采用iTRAQ技术结合LC-MS/MS，筛选两组差异蛋白，行 Mascot 数据库检索，Go在线对差异蛋白进行功能注释聚类分析，并运用KEGG对蛋白富集的信号通路进行分析。.结果：1.胃泌素刺激人结肠癌SW480细胞中CacyBP/SIP入核，构造CacyBP/SIP入核功能的细胞模型，蛋白免疫印迹验证CacyBP/SIP入核功能的细胞模型成功建立。2.激光共聚焦扫描显微镜术验证CacyBP/SIP入核功能的细胞模型成功建立。3. iTRAQ技术筛选出差异蛋白2136个，其中表达上调的有173个，表达下调的有132个。4.生物信息学分析表明，生物学过程的差异表达蛋白主要集中在生物合成过程（biosynthetic process）、细胞过程(cellular process)，分子功能分类中，差异表达蛋白主要富集于结合功能（ binding ）、催化功能（catalyti cactivity）。细胞成分(cellular component)的蛋白主要集中于organelle上。 筛选具有结合功能、定位于细胞核且差异倍数大于1.5的蛋白，共7个，分别为PML、VDAC2、AnnexinA1、UBE2、Filamin-A, Heat shock protein beta-1、Cathepsin B。Pathway 代谢通路注释筛选出67条通路，其中具有显著性的通路共6条，4条通路有关键蛋白表达..结论：iTRAQ筛选出CacyBP/SIP入核后的差异蛋白2136个，173个上调，132个下调。差异蛋白主要集中在生物合成及细胞过程，KEGG通路分析筛选出67条信号通路，P≤0.05的通路共6条；有意义蛋UBE2,VDAC2,AnnexinA1,Fila