IGF2BP1 promotes gastric cancer progression by stabilizing HS6ST2 mRNA in an N6-methyladenosine-dependent manner

Figure 1 Insulin-like growth factor 2 mRNA-binding protein 1 is highly expressed in gastric cancer and correlates with lymph node metastasis and poor prognosis. A: Immunohistochemical analysis showing higher N6-methyladenosine levels in gastric cancer tissues with lymph node metastasis (LNM) compared with those without LNM (n = 36); B: Analysis of GSE17187 indicating upregulation of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in gastric cancer (GC) tissues with LNM; C: Kaplan-Meier survival analysis of The Cancer Genome Atlas-stomach adenocarcinoma showing that high IGF2BP1 expression is associated with shorter overall survival; D: The Cancer Genome Atlas-stomach adenocarcinoma analysis demonstrating poorer overall survival in patients with GC with LNM; E: Immunohistochemical analysis confirming elevated IGF2BP1 expression in GC tissues relative to paired adjacent non-tumor tissues; F and G: IGF2BP1 mRNA and protein expression levels across GC cell lines, with MGC803 and SGC-7901 showing high expression. Data are presented as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. LN: Lymph node; m6A: N6-methyladenosine; IGF2BP: Insulin-like growth factor 2 mRNA-binding protein; TPM: Transcripts per million; HR: Hazard ratio; GAPDH: Gyceraldehyde-3-phosphate dehydrogenase.

Figure 2 Knockdown of insulin-like growth factor 2 mRNA-binding protein 1 inhibits the proliferation, migration, and invasion of gastric cancer cells in vitro. A and B: Quantitative reverse-transcription polymerase chain reaction analysis of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) knockdown efficiency at the mRNA level in MGC803 (A) and SGC-7901 (B) cells; C and D: Western blotting confirming IGF2BP1 knockdown at the protein level in MGC803 (C) and SGC-7901 (D) cells; E: Transwell assays showing the effects of IGF2BP1 knockdown on the migration and invasion abilities of MGC803 and SGC-7901 cells; F: Wound healing assays assessing the effects of IGF2BP1 knockdown on the migration ability of MGC803 and SGC-7901 cells; G: Cell counting kit-8 assay evaluating the effects of IGF2BP1 knockdown on cell proliferation in MGC803 and SGC-7901 cells. Data are presented as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. IGF2BP: Insulin-like growth factor 2 mRNA-binding protein; shRNA: Short hairpin RNA; NC: Negative control; GADPH: Gyceraldehyde-3-phosphate dehydrogenase; CCK-8: Cell counting kit-8.

Figure 3 Insulin-like growth factor 2 mRNA-binding protein 1 promotes tumor growth via an N6-methyladenosine-dependent mechanism. A and B: Subcutaneous xenograft tumor model was used to evaluate the effects of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) on tumor growth in vivo. Representative tumor images (A); tumor growth curves and final tumor weight statistics (B); C: Venn diagram of multi-omics analysis identifies heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) and SH3-domain binding protein 1 as core IGF2BP1 target genes linked to poor prognosis; D: Positive correlation between IGF2BP1 and HS6ST2 mRNA expression in The Cancer Genome Atlas-stomach adenocarcinoma; E: High HS6ST2 expression correlates with poor overall survival in The Cancer Genome Atlas-stomach adenocarcinoma; F: The expression of the HS6ST2 protein in gastric cancer tissues compared with adjacent non-tumor tissues; G: Predicted N6-methyladenosine (m6A) modification sites on HS6ST2 mRNA (sequence-based RNA adenosine methylation site predictor tool); H: M6A enrichment of HS6ST2 mRNA in SGC7901 and MGC803 cells (MeRIP-quantitative polymerase chain reaction); I and J: M6A levels of HS6ST2 mRNA are decreased upon IGF2BP1 knockdown (I) and increased upon its overexpression (J); K and L: IGF2BP1 overexpression increases the luciferase reporter activity driven by the wild-type HS6ST2 3’UTR (K), an effect that is lost upon mutation of the predicted m6A site (L). Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. IGF2BP: Insulin-like growth factor 2 mRNA-binding protein; shRNA: Short hairpin RNA; NC: Negative control; TPM: Transcripts per million; HR: Hazard ratio; HS6ST2: Heparan sulfate 6-O-sulfotransferase 2; m6A: N6-methyladenosine; RIP: RNA immunoprecipitation.

Figure 4 Heparan sulfate 6-O-sulfotransferase 2 promotes the proliferation, migration, and invasion of gastric cancer cells in vitro. A and B: Quantitative reverse-transcription polymerase chain reaction (A) and western blot (B) for the knockdown efficiency of heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) in SGC7901 cells at the mRNA and protein levels; C and D: Quantitative reverse-transcription polymerase chain reaction (C) and western blot (D) analyses of HS6ST2 overexpression efficiency in MGC803 cells at the mRNA and protein levels; E and F: Transwell (E) and Wound healing assay (F) for the effect of HS6ST2 knockdown on the invasion and migration ability of gastric cancer (GC) cells; G and H: Transwell (G) and Wound healing assay (H) for the effect of overexpression on the migration ability of GC cells; I and J: Cell counting kit-8 assay for the effect of HS6ST2 knockdown (I) and overexpression (J) on the proliferation of GC cells. Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. HS6ST2: Heparan sulfate 6-O-sulfotransferase 2; shRNA: Short hairpin RNA; GAPDH: Gyceraldehyde-3-phosphate dehydrogenase; NC: Negative control; OE: Overexpression.

Figure 5 Insulin-like growth factor 2 mRNA-binding protein 1 promotes the malignant phenotype of gastric cancer cells by regulating heparan sulfate 6-O-sulfotransferase 2. A and B: Western blot (A) and quantitative reverse-transcription polymerase chain reaction (B) for the expression levels of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) in SGC7901 cells after IGF2BP1 overexpression with or without HS6ST2 knockdown; C and D: Transwell (C) and wound healing (D) for the invasion and migration abilities of SGC7901 cells; E: Cell counting kit-8 assay for the proliferation ability of SGC7901 cells; F and G: Western blot (F) and quantitative reverse-transcription polymerase chain reaction (G) for the expression levels of IGF2BP1 and HS6ST2 in MGC803 cells after IGF2BP1 knockdown with or without HS6ST2 overexpression; H and I: Transwell (H) and wound healing (I) for the invasion and migration abilities of MGC803 cells; J: Cell counting kit-8 assay for the proliferation ability of MGC803 cells. Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. IGF2BP: Insulin-like growth factor 2 mRNA-binding protein; HS6ST2: Heparan sulfate 6-O-sulfotransferase 2; shRNA: Short hairpin RNA; NC: Negative control; GAPDH: Gyceraldehyde-3-phosphate dehydrogenase; CCK-8: Cell counting kit-8.

Figure 6 Heparan sulfate 6-O-sulfotransferase 2 suppresses apoptosis by activating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathway and is associated with the tumor microenvironment. A: Volcano plot of differentially expressed genes upon heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) knockdown (437 genes, 291 up, 146 down); B: Heatmap confirms distinct transcriptome profiles in control vs HS6ST2-knockdown groups; C: Kyoto Encyclopedia of Genes and Genomes pathway enrichment of differentially expressed genes shows significant enrichment in the phosphatidylinositol 3-kinase-protein kinase B signaling pathway; D: HS6ST2 knockdown significantly increases apoptosis in SGC7901 cells (flow cytometry); E: Western blot shows HS6ST2 knockdown reduces phosphorylation of phosphatidylinositol 3-kinase, protein kinase B and mammalian target of rapamycin; F: Immunohistochemical analysis reveals CD33 and periostin expression is significantly higher in gastric cancer tissues with lymph node metastasis. Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. KEGG: Kyoto Encyclopedia of Genes and Genomes; shRNA: Short hairpin RNA; NC: Negative control; P13K: Phosphatidylinositol 3-kinase; P: Phosphorylation; Akt: Protein kinase B; mTOR: Mammalian target of rapamycin; LN: Lymph node.