Fig. 5

POU6F2-AS1 tethers YBX1 to the FASN promoter to transcriptionally activate FASN and promotes CRC cell progression. (A) Venn diagram exhibiting the downstream genes coregulated by POU6F2-AS1 and YBX1, and overlapping analysis with lipid metabolism-related genes (LMGs). (B) qRT–PCR and Western blotting showed that the reduction in FASN in HCT116 cells with transfected with POU6F2-AS1 siRNAs was reversed by YBX1 overexpression, whereas the increase in FASN in LoVo cells transfected with POU6F2-AS1 overexpression plasmid was reversed by YBX1 knockdown. (C) The YBX1-binding motif and sites enriched in the FASN promoter was predicted by JASPAR website. (D, E) Luciferase reporter assays showed that the luciferase activity driven by the − 447/-136 fragment of the FASN promoter region was significantly reduced in HCT116 cells with YBX1 knockdown but enhanced in LoVo cells with YBX1 overexpression. (F) Luciferase reporter assays for − 447/-136 fragment of the FASN promoter activity in SW480 cells with POU6F2-AS1 knockdown with or without overexpressing YBX1 (left), and in LoVo cells with POU6F2-AS1 overexpression with or without knocking down YBX1 (right). (G) The schematic diagram of truncated FASN promoter. (H) ChIP-qPCR analysis was performed to determine the binding affinity of YBX1 to nine FASN promoter regions in SW480 cells, showing that YBX1 bound to P2-4 regions in the FASN promoter. ChIP-qPCR with IgG was used as the negative control. (I) ChIP-qPCR analysis for the enrichment of YBX1 at the FASN promoter regions in SW480 cells with PO6F2-AS1 knockdown. (J) Representative images showing the expression of FASN in CRC tissues and paired ANTs detected by IHC. (K) CRC patient-derived organoid infected with POU6F2-AS1 knockdown or FASN overexpression lentivirus. (L) CCK–8 assay in POU6F2-AS1 knockdown HCT116 cells with FASN overexpression. (M) Colony formation assay in POU6F2-AS1 knockdown HCT116 cells with FASN overexpression. (N) Oil Red O staining assay in POU6F2-AS1 knockdown HCT116 cells with FASN overexpression. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001