Fig 2: A graph illustrates that SCAP mediated GDF15-induced EMT of EC by regulating cholesterol metabolism. In addition, GDF15 was shown to be a direct target of miR-1324.

Fig 3: SCAP mediated GDF15-induced migration and invasion of ESCCs via cholesterol metabolism. (A) Expression levels of SREBF2 and HMGCR in SCAP-overexpressing GDF15-siRNA ESCCs determined by western blotting. (B–G) SCAP overexpression in GDF15-siRNA ESCCs induced cell migration, invasion, and EMT, which was reduced by β-CD treatment (2.5 mM). (H–J) Pretreatment with cholesterol (5 ug/ml) induced cell migration, invasion, and EMT of GDF15-siRNA ESCCs.

Fig 4: GDF15 is a direct target of miR-1324. (A) A schematic of the target sites (wild and mutant) of miR-1324 in the 3′ UTR of the GDF15 mRNA transcript. (B) After the transfection of miR-1324 mimics, miR-1324 expression in ESCCs was determined by RT-PCR. (C,D) Dual-Luciferase reporter assays performed in ESCCs co-transfected with 3′-UTR WT or MT plasmid with miR-1324 or negative control using Lipofectamine 2000. Relative luciferase activity was measured using the Dual-Luciferase® Reporter Assay System. (E) Expression of miR-1324 (green) in EC and adjacent non-tumor tissues were determined by FISH, nuclei were stained with DAPI (blue). (F) Subcellular co-localization analysis of the interaction between GDF15 (red) and miR-1324 (green) in EC tissues, nuclei were stained with DAPI (blue). (G) Expression levels of SCAP, SREBF2, and HMGCR determined by western blotting. (H–J) Migration and invasion of ESCCs after transfection with miR-1324 mimics were determined using transwell chamber assays. Expression levels of EMT-associated proteins E-cadherin, vimentin, snail, and slug were determined by western blotting. **P < 0.01.

Fig 5: SCAP mediated GDF15-induced lung metastasis of KYSE 150 via cholesterol metabolism. (A) Reduced lung metastasis was observed in mice transplanted with GDF15 knockdown KYSE 150 cells, which was reversed by SCAP overexpression or high cholesterol diet. Lung metastasis in transplanted SCAP overexpressing cells was partially reduced by intraperitoneal injection of β-CD (0.5 g/kg). (B) MiR-1324 expression in KYSE 150 cells transfected with lentivirus overexpressing miR-1324 determined by RT-PCR. (C) Mice injected with KYSE 150 cells transfected with lentivirus overexpressing miR-1324 had reduced lung metastasis. **P < 0.01.