在本研究中，我们首先使用 RT-PCR、qPCR 阐明了血清饥饿增强了 MG-63 细胞分化过程中人 GD3 合酶 (hST8Sia I) 基因和神经节苷脂 GD3 表达的水平以及骨形态发生蛋白 2 和骨钙素的表达。 、蛋白质印迹和免疫荧光显微镜检查。为了评估血清饥饿引起的 MG-63 细胞分化过程中 hST8Sia I 基因的上调，对 hST8Sia I 基因的启动子区域进行了功能分析。使用含有 hST8Sia I 基因各种构建体的荧光素酶报告分析系统进行的启动子分析证明，顺式作用区域位于 -1146/-646，其中包括已知转录因子 AP-1、CREB、c-Ets-1 和NF-κB 在 MG-63 细胞中响应血清饥饿时显示出最高水平的启动子活性。通过定点诱变、NF-κB 抑制和染色质证明，包含 NF-κB 结合位点的 -731/-722 区域对于 MG-63 细胞中血清饥饿表达 hST8Sia I 基因至关重要免疫沉淀 (ChIP) 测定。使用shRNA敲低hST8Sia I表明hST8Sia I和GD3的表达对MG-63细胞的分化没有明显影响。此外，血清饥饿引起的 hST8Sia I 基因转录激活受到 MG-63 细胞中 p38MAPK 抑制剂 SB203580 的强烈阻碍。这些结果表明，人骨肉瘤 MG-63 细胞中血清饥饿引起的 hST8Sia I 基因的转录活性受到 p38MAPK/NF-κB 信号通路的调节。版权所有：© 2023 An et al.这是一篇根据知识共享署名许可条款分发的开放获取文章，允许在任何媒体上不受限制地使用、分发和复制，前提是注明原始作者和来源。

In this study, we have firstly elucidated that serum starvation augmented the levels of human GD3 synthase (hST8Sia I) gene and ganglioside GD3 expression as well as bone morphogenic protein-2 and osteocalcin expression during MG-63 cell differentiation using RT-PCR, qPCR, Western blot and immunofluorescence microscopy. To evaluate upregulation of hST8Sia I gene during MG-63 cell differentiation by serum starvation, promoter area of the hST8Sia I gene was functionally analyzed. Promoter analysis using luciferase reporter assay system harboring various constructs of the hST8Sia I gene proved that the cis-acting region at -1146/-646, which includes binding sites of the known transcription factors AP-1, CREB, c-Ets-1 and NF-κB, displays the highest level of promoter activity in response to serum starvation in MG-63 cells. The -731/-722 region, which contains the NF-κB binding site, was proved to be essential for expression of the hST8Sia I gene by serum starvation in MG-63 cells by site-directed mutagenesis, NF-κB inhibition, and chromatin immunoprecipitation (ChIP) assay. Knockdown of hST8Sia I using shRNA suggested that expressions of hST8Sia I and GD3 have no apparent effect on differentiation of MG-63 cells. Moreover, the transcriptional activation of hST8Sia I gene by serum starvation was strongly hindered by SB203580, a p38MAPK inhibitor in MG-63 cells. From these results, it has been suggested that transcription activity of hST8Sia I gene by serum starvation in human osteosarcoma MG-63 cells is regulated by p38MAPK/NF-κB signaling pathway.Copyright: © 2023 An et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.