新型膜分子CMTM7在B细胞中的表达、功能和疾病相关性研究

CMTM (CKLF-like MARVEL transmembrane domain-containing family) is a novel gene family reported by the Department of Immunology, Peking University. In human, it is consisting of nine genes, CKLF and CMTM1-8. The structure and function of CMTM members are between classical chemokines and transmembrane-4 superfamily (TM4SF). They play important roles in the immune and male reproductive systems as well as tumorigenesis. CMTM7 is located at the tumor suppressor locus 3p22.3, which is frequently deleted in multiple tumors including esophageal carcinoma. CMTM7 is downregulated or silenced in esophageal carcinoma cell lines and primary tumors due to its methylation and high frequent LOH; ectopic CMTM7 expression in esophageal carcinoma cell lines induces G1 cycle arrest through upregulating p27 and downregulating CDK2 and CDK6; CMTM7 is located in cell membrane, it can promote EGFR internalization and further down regulate pAkt. The above work submitted to Cancer Res. is revised now. CMTM7 is highly expressed in immune cells, but its expression level is down regulated in the bone marrow cells of ALL（Acute Lymphoblastic Leukemia）and AML（Acute Myeloid Leukemia）patients. Japanese researcher reported that Cmtm7 interacts with Blnk in mouse B lymphoma cell line and negatively regulates BCR endocytosis. BLNK is an essential adaptor protein in BCR signaling pathway with significant roles in the development and differentiation of B cells; more importantly, BLNK also has tumor suppressor activities similar to CMTM7. In this project, we will investigate the expression profile of CMTM7 in B cells with different stages and different types; observe its function in the proliferation and activation of B cells; study the interacting site and functional relationship between CMTM7 and BLNK; analyze its physiological roles and its association with leukemia and lymphoma derived from B cells with Cmtm7 conditional knockout mice. In summary, this project is original and feasible with solid preliminary work.

CMTM是我室在国际上首次报道的新基因家族，CMTM7是其成员之一，定位于抑癌基因富集区3p22.3。甲基化修饰和LOH共同导致其在食管癌中表达下调；恢复CMTM7的表达可上调p27引起G1期阻滞，抑制细胞生长。CMTM7有膜定位形式，能促进EGFR内化，抑制Akt磷酸化。上述研究已投稿Cancer Res.，正在修稿中。CMTM7在免疫细胞中高表达，在ALL和AML患者的骨髓中表达下调；小鼠细胞中Cmtm7与Blnk相互作用，调控BCR内化。BLNK是BCR通路中的接头蛋白，在B细胞发育分化中起重要作用，并具有与CMTM7功能相似的抑癌基因特性。本项目将研究CMTM7在不同种类、不同发育阶段B细胞中的表达谱；分析其在B细胞增殖、活化中的功能和机制；明确CMTM7与BLNK的相互作用位点和功能关系；利用Cmtm7敲除小鼠研究CMTM7的生理功能及其与B细胞淋巴瘤、白血病的相关性。

CMTM是我室在国际上首次报道的新基因家族，CMTM7是其成员之一，在免疫系统高表达。小鼠CMTM7在IgM交联后能够与BLNK相互作用，促进其磷酸化。为研究CMTM7在免疫系统中的功能，我们构建了Cmtm7条件敲除小鼠（购买的ES 细胞，针对Cmtm7 外显子2-3），发现F1代Cmtm7 flox/+小鼠出生不久便死亡，利用过继转移实验，将14.5天的胎肝细胞转移至照射后的Rag1-/-小鼠体内，发现Cmtm7 flox/+小鼠B、T细胞发育正常，B2细胞的体外存活、增殖和Ig基因类别转换未受影响，但血清IgM下降，腹腔中B-1a细胞的比例和绝对数均显著下降，在LPS刺激下的增殖能力降低。之后我们重新制备了针对Cmtm7 外显子2-4的条件性基因敲除小鼠，已获得全身敲除小鼠（EIIa-Cre）和在B细胞中特异性敲除Cmtm7的纯合子小鼠（CD19-Cre），发现全身敲除Cmtm7不影响重要脏器及T、B细胞的发育，但腹腔中B-1a细胞的比例和绝对数明显下降，血清IgM明显下降，IgG无变化；在B细胞中特异性敲除Cmtm7也不影响B细胞发育，但腹腔中B-1a细胞和血清IgM明显下降，IgG无变化，与Cmtm7全身敲除小鼠、Cmtm7flox/+杂合子小鼠的表型一致。此外，CMTM7在慢性B细胞白血病中的表达显著下调，在Ramos细胞中稳定敲减CMTM7显著促进细胞增殖和存活，抑制细胞凋亡，细胞总IgM及膜表面IgM水平明显降低，但CD79B及CD19的表达没有变化。CMTM7调节BCR、EGFR等受体的表达和转运，但作用机制不清。目前对于EGFR转运的研究模型和通路相对成熟，因此，我们首先研究了CMTM7对EGFR转运及信号通路的影响，以便为BCR研究提供线索。我们通过系列实验发现CMTM7通过增强Rab5活性和早期内体融合，促进EGFR内化、降解，降低EGFR-Akt信号，证明CMTM7是一个新的Rab5活性调节分子。Rab5不仅参与EGFR转运，而且在BCR等免疫受体的运输和信号转导过程中也发挥重要作用，相关结果为深入研究CMTM7在B细胞中的作用机制奠定了基础。综上所述，CMTM7具有重要的免疫调节功能，在调控B-1a细胞的生存、BCR表达和B细胞肿瘤的发生发展中具有重要作用。