Fig 2: Effects of miR-215-5p overexpression on tumor growth in vivo and its involvement in CRC pathogenesis a Subcutaneously injected HCT-116+/+-miR-215-5p cells formed significantly smaller tumors compared with HCT-116+/+-control cells 25 days after application into NSG mice (n = 5). b Volume of in vivo formed tumors was significantly smaller in case of HCT-116+/+-miR-215-5p cells compared with HCT-116+/+-control cells. c Weight of in vivo formed tumors was significantly smaller in case of HCT-116+/+-miR-215-5p cells compared with HCT-116+/+-control cells. d Expression levels of miR-215-5p were significantly increased in HCT-116+/+-miR-215-5p tumors compared with HCT-116+/+-control tumors 25 days after initiation of the experiment. e Involvement of miR-215-5p in CRC pathogenesis—direct targets of miR-215-5p described first in this study are in red squares, direct targets of miR-215-5p described in previous studies are in brown. *P < 0.05, **P < 0.01, CTRL control cells, EMT epithelial–mesenchymal transition, XIAP X-chromosome-linked inhibitor of apoptosis, ZEB2 zinc-finger E-box-binding homeobox 2, DTL denticleless protein homolog, DLG5 discs large homolog 5, TYMS thymidylate synthase, HOXB9 homeobox protein HoxB9, EREG epiregulin

Fig 3: Elevated phosphorylation levels of SYK and LYN upregulate PGF to promote angiogenesis(A–C) The intersection of angiogenesis genes and key genes enriched in the pathway (A). (B) A protein-protein interaction (PPI) network of the top 15 key genes from (A) was constructed. Partial vascular-related key genes (C).(D) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in ccRCC cells (786-O and 769-P) treated with PD173952 (see Figure S9A for statistical graph). The data represent one of two independent experiments.(E) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in ccRCC cells (786-O and 769-P) treated with R406 (see Figure S9B for statistical graph). The data represent one of two independent experiments.(F) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in LYNY397E or LYNY397F ccRCC cells (786-O and 769-P) (see Figure S9C for statistical graph). The data represent one of two independent experiments.(G) Western blot of PDPK1, PIK3CA, SHH, EREG, and PGF in SYKY323/525E or SYKY323/525F ccRCC cells (786-O and 769-P) (see Figure S9D for statistical graph). The data represent one of two independent experiments.(H and I) Inhibited EREG or PGF in ABAT-knockdown ccRCC cells (786-O and 769-P) (see Figures S10A and S10B for statistical graph).(J) Tube formation assays using HUVECs cultured in CM from ABATsh PGFsi, ABATsh EREGsi, and ABATsh NCsi ccRCC cells (786-O and 769-P) treated with DMSO or sunitinib for 24 h (see Figures S10C and S10D for statistical graph).(K) Chick embryo chorioallantoic membrane (CAM) assays with conditioned medium from ABATsh PGFsi, ABATsh EREGsi, and ABATsh NCsi ccRCC cells (786-O and 769-P) treated with DMSO or sunitinib for 24 h (see Figures S10E–S10G for statistical graph). The data are presented as the means ± SEMs. Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test, two-way ANOVA with Tukey’s multiple comparisons test, or unpaired t test where appropriate. Each spot represents one subject. NC, negative control. ∗∗p < 0.01, ∗∗∗p < 0.0001, ∗∗∗∗p < 0.0001. See also Figures S9 and S10.

Fig 4: EREG and HOXB9 are direct targets of miR-215-5p a RT-qPCR analyses proved significantly reduced mRNA levels of EREG and HOXB9 in HCT-116+/+ cells. b RT-qPCR analyses proved significantly reduced mRNA levels of EREG and HOXB9 in DLD-1 cells. c RT-qPCR analyses proved significantly reduced mRNA levels of EREG and HOXB9 in HCT-116−/− cells. d RT-qPCR analyses proved significantly reduced mRNA levels of EREG and HOXB9 in HT-29 cells. e Luciferase assay confirmed EREG to be a direct target of miR-215-5p. f Luciferase assay confirmed HOXB9 to be a direct target of miR-215-5p. g Western blot analyses proved downregulated protein levels of EREG and HOXB9 in CRC cells transfected with miR-215-5p mimics. h Expression of EREG and HOXB9 is significantly upregulated in tumor tissues compared with non-tumor adjacent tissues. *P < 0.05, **P < 0.01, ***P < 0.001, CTRL control cells, HT healthy tissue, TT tumor tissue

Fig 5: A proposed model for the tumor-suppressive role of FTO in glioma. In normal glial cells or low-grade glioma, FTO is expressed and functions to demethylate EREG mRNA, leading to its degradation. This limits EREG expression, keeping the PI3K/Akt pathway in check and allowing p53/p21 to enforce cell cycle control, thereby suppressing proliferation. In high-grade glioma, FTO is downregulated. This leads to hypermethylation (↑m6A) and stabilization of EREG mRNA, resulting in EREG protein upregulation. The excess EREG activates the PI3K/Akt signaling cascade, which suppresses p53 and p21, leading to uncontrolled cell cycle progression and tumor growth.