Altern Ther Health Med. 2023 Nov;29(8):364-369.
Ovarian cancer is the leading cause of death linked to gynecological cancers. Notch1, as an important component of Notch signaling, plays an important role in a variety of cancers. This study aims to discuss the mechanisms through which Notch 1 influences the development of ovarian cancer.
To design and establish the short hairpin (sh) RNA for targeting Notch 1, we transfected THP-1 cells (one of the human macrophagic lines). The cells were divided into shRNA negative control (NC) group and the Notch 1 shRNA group. The CoC1 cells and THP-1 cells (human mononuclear macrophages) are co-cultured, which are injected into the nude mice subcutaneously based on proposition. The sizes of tumors and their volumes are observed through HE staining. Flow cytometry is used to sort out macrophages from subcutaneous tumors of nude mice, whose protein-related expression is detected through western blot. Then the NC group and the Notch 1 shRNA group in the co-culture system are treated with PI3K/mTOR Inhibitor-13 sodium (200 nM) for 48h and then co-cultured with human endothelial cell lines HUVEC, CoC1, and THP-1 to test the tube-forming capacity of HUVEC cells in each group to detect the protein-related expression in THP-1 cells using western blot.
It is seen that the Notch 1 shRNA group includes a significantly larger tumor size, decreased relative expression, and the obvious increase of the relative protein expression in p-PI3K, p-mTOR, HIF1α, and VEGF compared with the NC group. Through tube-forming experiments, the Notch1 shRNA group significantly increased the number of HUVEC tubes. However, after the use of PI3K/mTOR Inhibitor-13 sodium, the number of tubes decreased in the NC and Notch1 shRNA groups, and there is no significant discrepancy in comparison to the NC group. The in vitro western blotting results indicate no obvious variation of Notch 1's relative protein expression in both the NC group and Notch 1 shRNA group after the use of PI3K/mTOR Inhibitor-13 sodium, while the relative protein expression of p-PI3K, p-mTOR, HIF1α, and VEGF was significantly reduced and there was no significant difference.
This study found that specific knockout of Notch 1 in tumor-associated macrophages will promote the activation of the PI3K/mTOR signaling pathway and the expression of HIF1α and VEGF, thus promoting angiogenesis and the development of ovarian cancer. Thus, this study provides insight into novel prognostic biomarkers and therapeutic targets for the treatment and research of ovarian cancer.
卵巢癌是妇科癌症相关死亡的主要原因。Notch1 作为 Notch 信号的重要组成部分，在多种癌症中发挥重要作用。本研究旨在探讨 Notch1 影响卵巢癌发展的机制。
为了设计和建立靶向 Notch1 的短发夹（sh）RNA，我们转染了 THP-1 细胞（人巨噬细胞系之一）。细胞分为 shRNA 阴性对照（NC）组和 Notch1 shRNA 组。将 CoC1 细胞和 THP-1 细胞（人单核巨噬细胞）共培养，基于命题将其注入裸鼠皮下。通过 HE 染色观察肿瘤的大小和体积。通过流式细胞术从裸鼠皮下肿瘤中分离出巨噬细胞，通过 Western blot 检测其蛋白相关表达。然后用 PI3K/mTOR 抑制剂-13 钠（200 nM）处理共培养系统中的 NC 组和 Notch1 shRNA 组 48h，然后与人内皮细胞系 HUVEC、CoC1 和 THP-1 共培养，以测试各组 HUVEC 细胞的管形成能力，用 Western blot 检测 THP-1 细胞中的蛋白相关表达。
结果表明，与 NC 组相比，Notch1 shRNA 组肿瘤体积明显增大，相对表达量降低，p-PI3K、p-mTOR、HIF1α 和 VEGF 的相对蛋白表达明显增加。通过管形成实验，Notch1 shRNA 组显著增加了 HUVEC 管的数量。然而，在用 PI3K/mTOR 抑制剂-13 钠后，NC 组和 Notch1 shRNA 组的管数减少，与 NC 组相比无明显差异。体外 Western blot 结果表明，在用 PI3K/mTOR 抑制剂-13 钠后，NC 组和 Notch1 shRNA 组 Notch1 的相对蛋白表达无明显变化，而 p-PI3K、p-mTOR、HIF1α 和 VEGF 的相对蛋白表达明显降低，无显著差异。
本研究发现肿瘤相关巨噬细胞中 Notch1 的特异性敲除会促进 PI3K/mTOR 信号通路的激活以及 HIF1α 和 VEGF 的表达，从而促进血管生成和卵巢癌的发展。因此，本研究为卵巢癌的治疗和研究提供了新的预后生物标志物和治疗靶点。
