Fig 2: Working Model. Schematic representation of the mechanism through which GM-CSF increased the expression of miR-877-3p in gastric cancer cells, which suppressed the expression of SOCS2 as a target gene. SOCS2, as a suppressor gene of Jak2/Stat3 signaling, mediated the chemotherapy-induced cancer cell stemness and drug resistance.

Fig 3: GM-CSF treatment promoted cancer cell stemness in vitro and tumorigenesis in vivo. (A,C): Flow cytometry analysis of CD133 + CSCs in SGC7901 (A) and BGC823 (C) cells before and after stimulation with exogenous GM-CSF in the cell culture medium. B and D: Quantitative analysis of A (B) and C (D). (E,F): QRT-PCR analysis of the stemness genes expression (h-Tert, Klf4, Nanog and Oct4) in SGC7901 (E) and BGC823 (F) cells with or without treatment with GM-CSF. (G): Western blot analysis of the stemness genes expression in SGC7901 and BGC823 cells with or without treatment with GM-CSF. (H): A gastric cancer xenograft model by transplantation of SGC7901 cells with or without stimulation with GM-CSF into nude mice (n = 10 in each group). (I): Tumor growth curves in (H). (J): Tumor images in (H). (K): Tumor weight in (H). Data are presented as the mean ± SEM (N = 3 for in vitro assays, and N = 10 for in vivo assay). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: miR-877-3p activated Jak2/Stat3 signaling through targeting SOCS2 in gastric cancer. (A,B): RNA-seq analysis of the SGC7901 cells with or without stimulation with 5-FU (A) or GM-CSF (B) identified a list of differentially expressed downregulated genes. (C): 32 genes were overlapped from the 176 downregulated genes by 5-FU treatment, 207 downregulated genes by GM-CSF treatment, and 5,091 potential target genes of miR-877-3p predicted by TargetScan Human8.0, including SOCS2. (D,E): Western blot demonstrated inhibition of SOCS2 by miR-877-3p overexpression and promotion of SOCS2 by miR-877-3p knockdown in both SGC7901 and BGC823 cells. (F): Sequence alignment of wide type (WT) or miR-877-3p-binding sites-mutated (MU) 3'UTR of SOCS2. (G): luciferase reporter assay demonstrated inhibition of WT-SOCS2-3'UTR by miR-877-3p, but not MU-SOCS2-3'UTR. (H,I): Western blot demonstrated positive or negative regulation of p-Jak2 and p-Stat3 by overexpression or knockdown of miR-877-3p in both SGC7901 and BGC823 cells. (J): Western blot demonstrated downregulation of SOCS2 and activation of Jak2/Stat3 signaling by GM-CSF treatment in the tumors from the mice model. Data are presented as the mean ± SEM (N = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Induction of drug resistance and GM-CSF expression/secretion in the survived gastric cancer cells after chemotherapy. (A): IC50 analysis of the survived SGC7901 and BGC823 cells after treatment with 5-FU or DDP for 72 h. (B): QRT-PCR analysis of the GM-CSF mRNA levels in the survived SGC7901 and BGC823 cells. (C): Western blot analysis of the GM-CSF protein levels in the survived SGC7901 and BGC823 cells. (D): ELISA analysis of the GM-CSF levels in supernatants of the survived SGC7901 and BGC823 cells. Data are presented as the mean ± SEM (N = 3). *p < 0.05, **p < 0.01, ***p < 0.001.