MIER3通过MRGBP介导乙酰化修饰途径抑制结直肠癌侵袭转移的分子机制

Metastatic spreading is the main cause of cancer-related deaths. We previously demonstrated that MIER3 expression was significantly decreased in colorectal carcinoma (CRC), which was correlated with invasion, metastasis and clinical prognosis in patients with CRC. Up-regulation of MIER3 significantly inhibited proliferation, migration and invasiveness of CRC cells in vitro. Co-immunoprecipitation (CoIP) assays combined with mass spectrography revealed that MRGBP is a potentially direct interaction protein of MIER3 in CRC. Furthermore, it was demonstrated that the overexpression of MIER3 could suppress the expression of MRGBP and could change the acetylation modification for histone. Based on these findings, we hypothesize that MIER3 could inhibit MRGBP expression, and then induce some key genes by modifying acetylation state of histones, which mediating cell proliferation and metastasis in CRC. In this study, we will evaluate the function of MIER3 in CRC in vivo and in vitro by gain and loss of function model. Furthermore, we will explore the action way and motifs between MIER3 and MRGBP using CoIP assays, GST pull-down, and so on. Furthermore, the signaling molecules regulated by MIER3 and MRGBP are analyzed though gene expression microarrays，and establish a functional link among key molecules, MIER3 and MRGBP. The research will uncover the pathway of MIER3 effects on CRC, and will provide basic knowledge for future therapy of the MIER3 signaling pathway.

课题组前期研究发现MIER3在结直肠癌中表达下调，并与结直肠癌的侵袭、转移及临床预后相关，其过表达可抑制结直肠癌细胞的增殖、迁移及侵袭能力，并与结肠癌EMT密切相关。进一步，利用 CoIP结合质谱分析发现MIER3能与乙酰转移酶Tip/NuA4亚基MRGBP直接结合，通过抑制其表达影响乙酰化修饰作用。因此我们推测，MIER3可能通过MRGBP介导乙酰化修饰途径，调控某些关键基因的表达，进而调控结直肠癌的侵袭和转移。本项目拟利用RNAi、过表达等在体内、体外进一步明确MIER3在结直肠癌侵袭转移中的作用；CoIP、GST pull-down等验证确定MIER3和MRGBP的相互作用方式及结合域，在此基础上应用基因芯片分析MIER3与MRGBP 所共同调控的关键信号分子，明确它们内在功能联系，从而阐明一条完整的MIER3抑制结直肠癌侵袭转移的分子机制，为基于MIER3通路靶向治疗奠定理论基础。

MIER3是课题组应用基因表达谱从结直肠癌组织标本中筛选获得的一个候选结直肠癌转移抑制基因。实验表明其在结直肠癌中表达下调，并与结直肠癌转移及患者预后不良密切相关。我们应用过表达及干扰技术从体内、体外两方面功能实验证实MIER3的下调可显著增强结直肠癌细胞的增殖、侵袭及转移能力；其上调则可明显减低结直肠癌细胞的增殖、侵袭及转移能力，并与结肠癌EMT密切相关。进一步机制研究中我们利用蛋白双向电泳、免疫共沉淀结合蛋白质谱技术，筛选鉴定出MIER3 特异性结合蛋白MRGBP，以及PSME3和SP1两个重要的功能相关信号分子，并进行了相关验证工作；对它们在结直肠癌中的相关功能以及与MIER3 的相关性开展了研究，实验表明MRGBP与PSME3在结直肠癌中高表达，与淋巴结转移、远处转移、Dukes 分期、分化程度正相关，是影响结直肠癌预后的危险因素；机制实验证实MRGBP可通过激活Wnt信号通路，以及通过Tip60/NuA4介导的对NF-kb信号通路中p65的乙酰化修饰，促进结直肠癌细胞EMT，进而参与调控结直肠癌细胞的增殖、侵袭、迁移；而PSME3则可通过调控CyclinB1和p-cdc2蛋白表达，致使细胞发生G2/M期阻滞，进而增强结直肠癌细胞的放疗敏感性;此外，MIER3还可通过与SP1的相互作用，抑制结直肠癌细胞的EMT，抑制结直肠癌转移。本课题的实验结果证实MIER3在结直肠癌中是一重要的抑癌基因,MIER3可能为结直肠癌的治疗及预后判断潜在的新分子靶点，为阐明结直肠癌侵袭转移的相关机制提供了新的理论依据。