异粘蛋白MTDH在乳腺癌中通过PGK1提高糖代谢的机制研究

Breast cancer (BC) is the most frequently diagnosed cancer and the leading global cause of cancer deaths in women. Metabolism is the underline of the cancer’s growth, metastasis and drug resistance. There is no other research studies metadherin (MTDH) in glucose metabolism in cancer. In our previously study, we found that MTDH could promote cells to take more glucose into cells and increase cells to use glucose. From protein co-immunoprecipitation and mass spectral analysis, we found MTDH could interact phosphoglycerate kinase (PGK1), which is the key enzyme for glucose metabolism. We supposed that MTDH promoting glucose metabolism was through PGK1. Our study wants to perform protein immunoprecipitation, mass spectral analysis, the level of the metabolism molecules, enzymes’ activity and other experiments to study the mechanism of that MTDH through PGK1 increases cells’ glucose metabolism resulting in cancer cells proliferation, metastasis, chemo-resistance change. Through this study, it can replenish the mechanism of MTDH in cancer cells. Most of all is MTDH’s function in glucose metabolism. And it could lead us to understand the mechanism of breast cancer glucose metabolism more deeply.

乳腺癌是女性最常见的恶性肿瘤之一。代谢的改变影响着乳腺癌的生长、转移、耐药等各方面。我们在前期研究中发现异粘蛋白（metadherin，MTDH）可以提高细胞对葡萄糖的摄取及消耗。通过免疫共沉淀、质谱分析，发现MTDH可以与糖代谢中的关键酶磷酸甘油酸酯激酶1(phosphoglycerate kinase，PGK1)形成复合物，提示MTDH是通过影响PGK1进而影响肿瘤细胞葡萄糖代谢的。本课题拟通过蛋白相互作用、质谱分析、代谢相关分子检测、酶活性检测等试验来研究MTDH通过PGK1提高乳腺癌细胞葡萄糖摄取及消耗进而影响肿瘤增殖、转移、耐药等表型的机制研究。通过对该问题的研究可以对MTDH已知的分子机制进行补充，能够更加深入地阐明MTDH通过糖代谢对乳腺癌增殖、转移、耐药等机制的影响。进一步了解乳腺癌葡萄糖代谢改变的机制。

目的:异粘蛋白(Metadherin，MTDH)作为一种新的抗癌靶点，可通过调控多种途径促进肿瘤细胞的一系列恶性表型的出现。然而，MTDH在乳腺癌中糖代谢过程中的作用尚不清楚。本研究旨在探讨MTDH在乳腺癌细胞糖酵解中的作用及机制。.方法:利用肿瘤基因组图谱(the Cancer Genome Atlas，TCGA)数据库对MTDH mRNA表达谱进行差异分析。通过转录组测序分析MTDH的生物学功能。采用超高效液相色谱-串联质谱(UPLC-MS/MS)对非靶向代谢组学进行分析。测定MTDH过表达细胞系的葡萄糖摄取率、葡萄糖-6-磷酸(G6P)、NADPH/NADP+比值、乳酸脱氢酶( lactate dehydrogenase，LDH)含量和乳酸产量。通过免疫沉淀和蛋白质谱检测糖酵解与MTDH相互作用的关键酶蛋白，并结合蛋白共免疫沉淀和western blotting（WB）进一步验证。.结果:MTDH在乳腺癌组织中高表达，且MTDH在乳腺癌组织中的表达高于癌旁组织。转录组测序显示MTDH参与细胞内葡萄糖代谢。UPLC-MS/MS结果进一步表明MTDH在乳腺癌细胞糖酵解中起重要作用，特别是在丙酮酸代谢中。进一步鉴定MTDH的过表达可以促进乳腺癌细胞对葡萄糖的摄取和利用、G6P含量、LDH活性、乳酸生成以及NADP+向NADPH的转化。质谱和后期WB验证了MTDH与PGK1相互作用。MTDH不改变PGK1在mRNA和蛋白水平上的表达，但MTDH可与乳腺癌细胞中的磷酸甘油酸激酶1 (Phosphoglycerate kinase 1，PGK1)相互作用。通过构建MTDH的截短体和蛋白免疫沉淀，进一步发现PGK1可能与MTDH的259-363区结合。在MTDH稳定过表达的BC细胞中，敲低PGK1后一系列糖酵解相关指标降低。.结论:MTDH在乳腺癌细胞糖酵解过程中起重要作用，且与PGK1相互作用可促进这一过程。MTDH可通过PGK1参与糖酵解和细胞转移。