Department of Medical Oncology, Yantaishan Hospital, Yantai, Shandong, China.
Department of Oncology, Harbin Chest Hospital, Harbin, Heilongjiang, China.
J Biol Regul Homeost Agents. 2019 Jul-Aug;33(4):1085-1095.
The purpose of this study was to explore the effect of Allograft Inflammatory Factor 1 (AIF-1) on the regulation of proliferation of breast cancer cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), cell culture and counting, and mass spectrometry were performed. The biologically active high-purity recombinant protein rhAIF-1 was obtained by optimizing the rhAIF-1 protein purification system, and MDA-MB-231 and MDA-MB-361 breast cancer cell lines were used. After adding to the culture medium, rhAIF-1 was found to promote cell proliferation in dose-dependent and time-dependent manners. The purified protein rhAIF-1 was marked with rhodamine and incubated with the cells. Confocal imaging analysis revealed that the foreign protein was localized in the cytoplasm, and rhAIF-1 was unevenly distributed in the cytoplasm. Although AIF-1 accumulates around the nucleus, it can not enter the nucleus, suggesting that other factors might be involved in the regulation of cell proliferation. In order to find the possible interacting protein of rhAIF-1, protein immunoprecipitation technique and mass spectrometry were employed, and it was indicated that ADAM28m was the possible interacting protein of rhAIF-1. The interaction between rhAIF-1 and ADAM28m was validated by immunoprecipitation along with Western blotting. It was found that rhAIF-1 could precipitate ADAM28m protein by immunoprecipitation. The results indicated that IF-1 participates in the development of breast cancer by interacting with ADAM28m and activating downstream signaling pathways. It was concluded that AIF-1 provides a new idea for the molecular mechanism of breast cancer cell proliferation and acts as a new target for the prevention and treatment of breast cancer in the future.
本研究旨在探讨异柠檬酸脱氢酶 1（AIF-1）在调节乳腺癌细胞增殖中的作用。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、细胞培养和计数、质谱等方法，优化 rhAIF-1 蛋白纯化体系，获得具有生物学活性的高纯度重组蛋白 rhAIF-1，并用 MDA-MB-231 和 MDA-MB-361 乳腺癌细胞系进行实验。结果发现，rhAIF-1 可促进细胞增殖，且具有浓度和时间依赖性。用 rhodamine 标记纯化蛋白 rhAIF-1 并与细胞孵育，共聚焦成像分析显示，该外源性蛋白定位于细胞质中，且 rhAIF-1 在细胞质中不均匀分布。虽然 AIF-1 在核周聚集，但不能进入细胞核，提示可能有其他因素参与细胞增殖的调控。为了寻找 rhAIF-1 的可能相互作用蛋白，采用蛋白质免疫沉淀技术和质谱分析，发现 ADAM28m 是 rhAIF-1 的可能相互作用蛋白。免疫沉淀结合 Western blot 验证了 rhAIF-1 与 ADAM28m 的相互作用。免疫沉淀结果显示 rhAIF-1 可沉淀 ADAM28m 蛋白。结果表明，IF-1 通过与 ADAM28m 相互作用并激活下游信号通路，参与乳腺癌的发生发展。综上所述，AIF-1 通过与 ADAM28m 相互作用，为乳腺癌细胞增殖的分子机制提供了新的思路，有望成为未来乳腺癌预防和治疗的新靶点。
