Fig 2: Placental growth factor (PLGF) promotes gallbladder cancer (GBC) cell metastasis and stemness in vitro. A, Quantitative RT-PCR analysis of PLGF levels in H69, 293T, NOZ, and GBC-SD cells. B, Immunoblotting analyses of the protein levels of PLGF in H69, 293T, NOZ, and GBC-SD cells. C, Quantitative RT-PCR analysis of PLGF levels in NOZ cells after transfection with PLGF siRNA (si-1, si-2, and si-3) or negative control siRNA (si-NC). ***P < .001. D-F, Knockdown of PLGF suppresses NOZ cell migration and invasion. Exogenous PLGF reverses this process in GBC-SD cells. Scale = 100 µm. D, Representative images. E, Quantitative analysis of the number of migrated and invaded NOZ cells after treatment with siRNA. ***P < .001. F, Quantitative analysis of the number of migrated and invaded GBC-SD cells after treatment with exogenous PLGF. ***P < .001. Data are presented as the mean ± SD. G, Immunoblotting analyses of the levels of epithelial–mesenchymal transition marker proteins in treated GBC cells. E-cad, E-cadherin; N-cad, N-cadherin. H, Immunofluorescence analysis of Slug and N-cadherin in treated GBC-SD cells. Scale = 100 µm. I, Analysis of the sphere-forming potential of GBC cells after treatment with or without 100 ng/mL PLGF. Shown are representative light microscopy images. Scale bar = 100 µm. J, Quantitative analysis of the numbers of spheres. Only spheres >50 µm in diameter were counted. **P < .01. K, Immunoblotting analyses of the levels of cancer stem cell marker proteins in treated GBC cells

Fig 3: Gene set enrichment analysis (GSEA) of hub genes in the TCGA-LIHC MAPK-RAP1A dataset. (A–F) Top three gene sets (according to GSEA enrichment score) enriched in the high-expression group of single hub genes. (A) PGF, (B) STMN1, (C) ANGPT2, (D) FLT3, (E) HSPA8, and (F) RAP1A.

Fig 4: High placental growth factor (PLGF) expression correlates with poor clinical outcomes in patients with gallbladder cancer (GBC). A, Western blot analysis of PLGF protein levels in 10 paired GBC (T) and normal adjacent tissue (N) samples. ß-Actin was used as a loading control. B, Scatterplots of the relative expression levels of PLGF in another 39 paired GBC tissues and their corresponding normal adjacent tissues. PLGF expression was calculated and is expressed as the PLGF/GAPDH expression ratio (2-? CT); P = .0477. C, Kaplan–Meier overall survival curve of GBC patients based on PLGF expression; P = .0375

Fig 5: MicroRNA‐19a (miR‐19a) knockdown attenuates the promotion effects of placental growth factor (PLGF) on gallbladder cancer (GBC) cells. A, B, Migration and invasion assays of GBC‐SD cells infected with antagomir‐19a lentivirus or negative control (antagomir‐NC) and treated with or without 100 ng/mL PLGF. A, Representative images. B, Quantitative analysis of the numbers of migrated and invaded cells. C, D, Sphere‐formation assays of GBC‐SD cells infected with antagomir‐19a lentivirus or antagomir‐NC and treated with or without 100 ng/mL PLGF. (C) Representative images. D, Quantitative analysis of the numbers of spheres. *P < .05; **P < .01. E, Immunoblotting analyses of the levels of epithelial–mesenchymal transition (EMT) and cancer stem cell marker proteins in GBC‐SD cells after infection with antagomir‐19a lentivirus or antagomir‐NC and treatment with or without 100 ng/mL PLGF. E‐cad, E‐cadherin; N‐cad, N‐cadherin. F, Working model of the PLGF–c‐Myc–miR‐19a–PTEN regulatory axis in GBC