Department of Clinical Laboratory, Hunan Cancer Hospital &The affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, 410013, China.
Department of Clinical Laboratory, Zhuhai Hospital, Jinan University, Zhuhai, 519000, Guangdong, China.
J Exp Clin Cancer Res. 2018 Nov 26;37(1):284. doi: 10.1186/s13046-018-0956-3.
MNAT1 (menage a trois 1, MAT1), a cyclin-dependent kinase-activating kinase (CAK) complex, high expresses in various cancers and is involved in cancer pathogenesis. However, mechanisms underlying its regulation in carcinogenesis are unclear.
The tissue microarray of colorectal cancer (CRC) was used to evaluate MNAT1 expressions in CRC tissues using immunohistochemistry, CRC cell lines were also detected MNAT1 expression using Western-blotting. MNAT1 and shMNAT1 vectors were constructed, and transfected into CRC cells. Cell growths of the transfected cells were observed using MTT and colony formation. The affects of MNAT1 on p53 expression were analyzed using Western-blotting and Real-time PCR. Immunoprecipitation assay was used to analyze the interaction p53 and MNAT1, and Western-blotting was used to test the effects of MNAT1 on p53 downstream molecules. The apoptosis of CRC cells with MNAT1 or shMNAT1 were analyzed using flow cytometry. BABL/c athymic nude mice were used to observe the effect of MNAT1 on CRC cell growth in vivo.
MNAT1 was found to be overexpressed in CRC tissues and cells, and MNAT1 expressions in CRC tissue samples were associated with CRC carcinogenesis and poor patient outcomes. MNAT1-knockin increased CRC cell growth and colony formation, and MNAT1-knockdown dramatically decreased cell motility and invasion. MNAT1 physically interacted with p53, MNAT1 also increased the interaction of MDM2 with p53. Strikingly, MNAT1 mediated p53 ubiquitin-degradation. MNAT1 shortened p53 half-life, and ectopic MNAT1 expression decreased p53 protein stability. Moreover, MNAT1 induced RAD51 and reduced p21, cleaved-caspase3, cleaved-PARP and BAX expression. MNAT1 inhibited CRC cell apoptosis. shMANT1 decreased tumor growths in nude mice following p53 increase.
MNAT1 binds to p53, mediates p53 ubiquitin-degradation through MDM2, increases cell growth and decreases cell apoptosis, and finally promotes CRC malignance. MNAT1 binding to p53 and mediating p53 ubiquitin-degradation axis represents a novel molecular joint in the p53 pathway.
MNAT1（三人行 1，MAT1）是一种细胞周期蛋白依赖性激酶激活激酶（CAK）复合物，在各种癌症中高表达，并参与癌症发病机制。然而，其在致癌作用中的调节机制尚不清楚。
使用免疫组织化学法检测结直肠癌（CRC）组织中的 MNAT1 表达，使用 Western-blotting 检测 CRC 细胞系中的 MNAT1 表达。构建 MNAT1 和 shMNAT1 载体，并转染 CRC 细胞。使用 MTT 和集落形成实验观察转染细胞的细胞生长。使用 Western-blotting 和实时 PCR 分析 MNAT1 对 p53 表达的影响。使用免疫沉淀实验分析 p53 和 MNAT1 的相互作用，使用 Western-blotting 检测 MNAT1 对 p53 下游分子的影响。使用流式细胞术分析具有 MNAT1 或 shMNAT1 的 CRC 细胞的凋亡。使用 BABL/c 无胸腺裸鼠观察 MNAT1 对 CRC 细胞生长的体内影响。
发现 MNAT1 在 CRC 组织和细胞中过度表达，CRC 组织样本中的 MNAT1 表达与 CRC 癌变和患者预后不良相关。MNAT1 敲入增加 CRC 细胞生长和集落形成，而 MNAT1 敲低则显著降低细胞迁移和侵袭。MNAT1 与 p53 发生物理相互作用，MNAT1 还增加了 MDM2 与 p53 的相互作用。引人注目的是，MNAT1 介导了 p53 的泛素降解。MNAT1 缩短了 p53 的半衰期，并且外源性 MNAT1 表达降低了 p53 蛋白稳定性。此外，MNAT1 诱导 RAD51 并降低了 p21、cleaved-caspase3、cleaved-PARP 和 BAX 的表达。MNAT1 抑制了 CRC 细胞凋亡。shMANT1 增加 p53 后，降低了裸鼠中的肿瘤生长。
MNAT1 与 p53 结合，通过 MDM2 介导 p53 泛素降解，增加细胞生长并减少细胞凋亡，最终促进 CRC 恶变。MNAT1 与 p53 的结合及其介导的 p53 泛素降解轴代表了 p53 途径中的一个新的分子结合点。
