Department of Pathology, Xiangya Hospital, Central South University, Changsha, 410008, Hunan Province, People's Republic of China.
Department of Gynecology, Xiangya Hospital, Central South University, Changsha, 410008, Hunan Province, People's Republic of China.
Mol Cell Biochem. 2022 Nov;477(11):2493-2505. doi: 10.1007/s11010-022-04436-x. Epub 2022 May 19.
This study aimed to investigate the role of cancer-associated fibroblast (CAF)-derived midkine (MK) in cisplatin (DDP) resistance. The primary cultures of CAFs and non-cancer fibroblasts (NFs) were isolated and purified. The DDP-resistant gastric cancer (GC) cells were cultured with CAF-conditioned medium. QRT-PCR and Elisa assays were employed to determine MK expression. The expression of ST7-AS1 was measured by qRT-PCR. The impact of CAFs, MK, and ST7-AS1 silencing on DDP resistance was determined by MTT and Annexin V/PI staining assay. Expression of EMT markers and PI3K/AKT was determined by Western blot and qRT-PCR. The role of MK in DDP resistance was confirmed in a xenograft model. Incubation with CAF-conditioned medium increased the IC50 to DDP. Also, incubation with CAF-conditioned medium increased cell viability, reduced cell apoptosis, and promoted EMT in DDP-resistant GC cells, which were all blocked with MK neutralization antibody treatment. MK increased the DDP resistance and upregulated the expression of ST7-AS1 in DDP-resistant GC cells. Additionally, ST7-AS1 knockdown increased the sensitivity to DDP by inhibiting EMT. Moreover, ST7-AS1 knockdown significantly decreased the phosphorylation of PI3K and AKT, and suppressed EMT, which were restored by MK addition. Finally, MK promoted tumor growth and DDP resistance in a mice model bearing the SGC-7901/DDP xenografts. CAF-derived MK promotes EMT-mediated DDP resistance via upregulation of ST7-AS1 and activation of PI3K/AKT pathway.
本研究旨在探讨癌相关成纤维细胞（CAF）衍生的中期因子（MK）在顺铂（DDP）耐药中的作用。原代培养 CAF 和非癌成纤维细胞（NF）并进行分离和纯化。用 CAF 条件培养基培养 DDP 耐药胃癌（GC）细胞。采用 QRT-PCR 和 Elisa 检测 MK 表达。采用 qRT-PCR 检测 ST7-AS1 的表达。通过 MTT 和 Annexin V/PI 染色实验确定 CAFs、MK 和 ST7-AS1 沉默对 DDP 耐药的影响。通过 Western blot 和 qRT-PCR 检测 EMT 标志物和 PI3K/AKT 的表达。MK 在 DDP 耐药中的作用在异种移植模型中得到了验证。用 CAF 条件培养基孵育增加了 DDP 的 IC50。此外，用 CAF 条件培养基孵育增加了 DDP 耐药 GC 细胞的活力，减少了细胞凋亡，促进了 EMT，而用 MK 中和抗体处理则阻断了这一作用。MK 增加了 DDP 耐药性，并上调了 DDP 耐药 GC 细胞中 ST7-AS1 的表达。此外，ST7-AS1 敲低通过抑制 EMT 增加了对 DDP 的敏感性。此外，ST7-AS1 敲低显著降低了 PI3K 和 AKT 的磷酸化，并抑制了 EMT，而 MK 的加入则恢复了这些作用。最后，MK 在 SGC-7901/DDP 异种移植瘤荷瘤小鼠模型中促进了肿瘤生长和 DDP 耐药。CAF 衍生的 MK 通过上调 ST7-AS1 和激活 PI3K/AKT 通路促进 EMT 介导的 DDP 耐药。
