Fig 2: AFP regulates the migration and invasion of cells through the PTEN/AKT1/SOX5/CES1 signaling axis(A) The JASPAR, ALGGEN and Genomstix databases were used to jointly predict the transcription factors that regulate CES1, and seven genes (TFAP2A, ZEB1, SOX2, SOX5, SOX9, SP1, PRRX2) that may be involved in regulating the expression level of CES1 were identified through literature screening. (B) RT‒PCR results revealed that knockdown of AFP significantly inhibited TFAP2A, SOX2, SOX5 and PRRX2. (C) TFAP2A, SOX2, SOX5 and PRRX2 were knocked down individually, and knockdown of TFAP2A, SOX2 and SOX5 significantly inhibited CES1 expression. (D-E) The results of the cell migration test revealed that knockdown of SOX5 significantly inhibited cell migration and invasion, while knockdown of TFAP2A and SOX2 did not; (F) knockdown of AFP also significantly inhibited the expression of SOX5 protein. The above results suggest that AFP may inhibit cell migration and invasion through the SOX5/CES1 signaling axis. (G) AFP may affect tumor cell migration and invasion through the PTEN/AKT1/SOX5/CES1 signaling axis in AFPP-GC. * P<0.05, ** P<0.01, ** P<0.001.

Fig 3: Enhancers and repressors of each organoid in HM and DM, as shown by the integrated analysis of DEGs and DARs at the bulk level(A and B) Strategy to identify enhancers and repressors differing in liver organoids treated with HM and DM at the bulk level. (B) Gene expression and scATAC-seq peaks were divided into DEGs and DARs of organoids treated with HM and DM at the bulk level, with OCRs associated with a specific gene defined as having an intersection score of p < 0.05 (left). The four patterns of regulation of gene expression in liver organoids treated with HM and DM (right). DM enhancer: both bulk-DEG and bulk-DAR increased in DM; DM repressor: bulk-DEG increased in DM, but bulk-DAR decreased in HM; HM repressor: bulk-DEG increased in HM, but bulk-DAR increased in DM; HM enhancer: both bulk-DEG and bulk-DAR increased in HM. The pair type counts were based on the number of pairs between OCRs related to the genes.(C) Gene-OCR pairs determined by log2(fold change) of scRNA-seq and scATAC-seq at the bulk level.(D) Representative TFs summarized for each pair type.(E) Genomic browser showing the bulk-level scATAC-seq for OCRs corresponding to enhancers and repressors of the AFP gene (chr4:74,273,152-74,328,858).(F) Log2(fold change) of the bulk-level DAR of OCRs corresponding to Figure 5E.(G) Interaction score (top) and enhancer and repressor TF (bottom) in the OCRs corresponding to Figure 5E.

Fig 4: miR-1270/AFP regulatory loop plays an important role in cell biological Behavior. (A) Western blot analysis of AFP level in HepG2 cells transfected with miR-1270 inhibitor (with or without AFP shRNA). GAPDH was used as a control. (B) Western blot analysis of AFP level in SMMC-7721 cells transfected with miR-1270 mimics (with or without AFP overexpression vector). (C) EdU assay showed proliferation of HepG2 cells. (D) EdU assay showed proliferation of SMMC-7721 cells. (E) Transwell assay showed migration of HepG2 cells. (F) Transwell assay showed migration of SMMC-7721 cells. Results were presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001. versus control group. All of the experiments were performed in triplicate.

Fig 5: CES1 may be a potential downstream target of AFP regulating cell migration and invasion in AFPP-GC.(A) Transcriptome sequencing analysis of genes differentially expressed after knockdown of AFP expression. (B) A total of 22 potential downstream targets were identified through the intersection of significant differentially expressed genes in AFPP-GC tissues vs. adjacent normal tissues, AFPP-GC samples vs. AFPN-GC samples, and si-NC samples vs. si-AFP samples. (C) The RT‒PCR results showed that the RNA expression levels of PANX2, SERPINC1, CES1 and CFB were significantly reduced in the si-AFP group. (D) Cell migration tests revealed that the expression of SERPINC1 and CES1 was significantly related to the migration ability of FU97 cells. (E) The RNA expression levels of SERPINC1 and CES1 were significantly higher in AFPP-GC than in normal adjacent tissues by tissue PCR. CES1 expression was significantly higher in AFPP-GC than in AFPN-GC, while SERPINC1 expression showed no significant difference between the two groups. (F) Western blot detection showed that knockdown of AFP expression significantly inhibited the expression of CES1 protein. (G) The results of the cell invasion test revealed that knockdown of CES1 could significantly inhibit the invasion of cells. (H) The rescue test revealed that knockdown of CES1 expression significantly inhibited the ability of AFP overexpression to promote cell migration. * P<0.05, ** P<0.01, ** P<0.001.