Resveratrol suppresses ulcerative colitis and colitis-associated colorectal cancer via modulating the miR-31-5p/SATB2 axis

Figure 1 MiR-31-5p expression levels are elevated in human colorectal cancer, human ulcerative colitis, mouse colitis-associated colorectal cancer, and HCT116 colorectal cancer cells. A: The Cancer Genome Atlas-derived box plot of miR-31-5p expression in colorectal cancer tissues vs normal controls. ^cP < 0.001 vs normal controls; B: Heatmap of differentially expressed microRNAs in a pilot human ulcerative colitis (UC) vs adjacent normal controls; C: Volcano plot of differentially expressed microRNAs in a pilot human UC vs adjacent normal controls; D: MiR-31-5p expression in paired inflamed and adjacent normal controls colonic tissues from pilot UC patients. ^aP < 0.05 vs adjacent normal controls (n = 4 pairs); E: Quantitative reverse transcription polymerase chain reaction quantification of colonic miR-31 in azoxymethane/dextran sulfate sodium-treated C57BL/6 mice. ^bP < 0.01 vs control (n = 7); F: Quantitative reverse transcription polymerase chain reaction quantification of miR-31-5p expression in NCM-460 cells and HCT116 cells. ^bP < 0.01 vs NCM-460 (n = 7). N: Normal controls; UC: Ulcerative colitis; AOM: Azoxymethane; DSS: Dextran sulfate sodium.

Figure 2 MiR-31-5p modulates migration, proliferation, apoptosis, and related protein expression in HCT116 colorectal cancer cells. A: Quantitative reverse transcription polymerase chain reaction analysis of miR-31-5p expression in HCT116 cells transfected with miR-31-5p mimic or inhibitor (n = 3); B: Wound healing assay showing the effect of miR-31-5p on HCT116 cell migration (scale bar = 200 μm) (n = 3); C: Colony formation assay showing the effect of miR-31-5p on HCT116 cell proliferation (n = 4); D: Flow cytometric analysis of the effect of miR-31-5p on HCT116 cell apoptosis (n = 3); E: Western blotting analysis of the effect of miR-31-5p on apoptosis-related protein expression in HCT116 cells (n = 3). ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^eP < 0.01 vs miR-31-5p mimic (30 nM), ^hP < 0.01 vs miR-31-5p inhibitor (50 nM).

Figure 3 MiR-31 overexpression exacerbates dextran sulfate sodium-induced colitis in mice. A: Schematic timeline of dextran sulfate sodium (DSS)-induced colitis in miR-31 transgenic mice; B: Body weight changes with colitis induction. ^bP < 0.01 vs control, ^dP < 0.05 vs DSS; C: Representative hematoxylin and eosin-stained colonic sections from each group (scale bar = 50 μm, 100 μm); D: Histopathological injury scores. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^eP < 0.01 vs DSS; E: Colon length measurements. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^dP < 0.05 vs DSS; F: Spleen index (spleen weight/body weight). ^bP < 0.01 vs control, ^eP < 0.01 vs DSS; G: Quantitative reverse transcription polymerase chain reaction analysis of miR-31 expression in colonic tissues. ^aP < 0.05 vs control, ^dP < 0.05 vs DSS; H: Representative western blots and quantification of inflammatory protein expression levels (cyclooxygenase-2, iNOS, tumor necrosis factor-α) in colonic tissues. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^eP < 0.01 vs DSS (n = 5); I: Representative western blots and quantification of mitochondrial apoptosis-related protein expression levels (cytochrome C, caspase-9, caspase-3) in colonic tissues. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^dP < 0.05 vs control, ^eP < 0.01 vs DSS (n = 5); J: Representative immunohistochemistry images and semi-quantitative analysis of Toll-like receptor 4, nuclear factor-κB, cyclooxygenase-2, and tumor necrosis factor-α expression levels (scale bar = 100 μm, n = 3). ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^dP < 0.05 vs DSS, ^eP < 0.01 vs DSS; K: Representative terminal deoxynucleotidyl transferase dUTP Nick End Labeling staining of apoptotic cells in colonic tissues (scale bar = 50 μm) and quantitative analysis of the apoptotic index. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^eP < 0.01 vs DSS (n = 3). NS: Not significant; DSS: Dextran sulfate sodium; COX-2: Cyclooxygenase-2; TNF-α: Tumor necrosis factor-α; Cyt-C: Cytochrome C; TLR-4: Toll-like receptor 4; NF-κB: Nuclear factor-κB; IHC: Immunohistochemistry.

Figure 4 MiR-31-5p targets SATB2, and this targeting relationship is functionally relevant in ulcerative colitis and colorectal cancer. A: Venn diagram showing shared hsa-miR-31-5p target genes predicted by miRTarBase, miRWalk, and TargetScan; B: Quantitative reverse transcription polymerase chain reaction analysis of SATB2 and PPP2R2A mRNA levels in HCT116 cells transfected with miR-31-5p mimic or inhibitor. ^aP < 0.05 vs control, ^bP < 0.01 vs control (n = 3); C: Schematic representation of wild-type and mutant luciferase reporter constructs containing the miR-31-5p binding site in the 3’-untranslated region of SATB2; D: Dual-luciferase reporter assay confirming that miR-31-5p directly targeted SATB2 but not PPP2R2A. ^bP < 0.01 vs NC + SATB2-wild-type (n = 3); E: Western blot analysis of SATB2 protein expression in HCT116 cells following miR-31-5p modulation. ^aP < 0.05 vs control, ^bP < 0.01 vs control (n = 4); F-H: Heatmap, volcano plot, and box plot showing differentially expressed mRNAs and SATB2 downregulation in human ulcerative colitis inflamed vs adjacent control tissues. ^aP < 0.05 vs N (n = 4 pairs); I: Correlation analysis between miR-31-5p and SATB2 expression in human ulcerative colitis tissues, r = -0.9; J-L: Validation of SATB2 expression in HCT116 colorectal cancer cells and dextran sulfate sodium-induced colitis in miR-31 transgenic mice by quantitative reverse transcription polymerase chain reaction, western blot, and immunohistochemistry. J, ^bP < 0.01 vs NCM-460; K and L: ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^dP < 0.05 vs dextran sulfate sodium, ^eP < 0.01 vs dextran sulfate sodium; M: Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses of SATB2-associated downstream genes. KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene Ontology; DSS: Dextran sulfate sodium; UC: Ulcerative colitis.

Figure 5 SATB2 partially counteracts miR-31-5p-driven malignant phenotypes in HCT116 cells. A: Quantitative reverse transcription polymerase chain reaction identification of the optimal si-SATB2 concentration in HCT116 cells. ^bP < 0.01 vs control, ^eP < 0.01 vs si-SATB2 (3 nM), ^hP < 0.01 vs si-SATB2 (5 nM) (n = 3); B: Western blot validation of SATB2 overexpression efficiency following 200 nM OE-SATB2 plasmid transfection. ^bP < 0.01 vs control (n = 4); C: Wound healing assays showing partial reversal of miR-31-5p-enhanced HCT116 cell migration by SATB2, (scale bar = 200 μm). ^aP < 0.05 vs NC + vector, ^dP < 0.05 vs vector + mimic, ^gP < 0.05 vs NC + OE-SATB2 (n = 3); D: Colony formation assays demonstrating attenuation of miR-31-5p-induced HCT116 cell proliferation by SATB2. ^bP < 0.01 vs NC + vector, ^dP < 0.05 vs vector + mimic, ^hP < 0.01 vs NC + OE-SATB2 (n = 3); E: Flow cytometric analysis of HCT116 cells apoptosis indicating partial rescue of miR-31-5p-mediated anti-apoptotic effects by SATB2. ^bP < 0.01 vs NC + vector, ^eP < 0.01 vs vector + mimic, ^hP < 0.01 vs NC + OE-SATB2 (n = 3); F: Western blotting of inflammatory signaling proteins (Toll-like receptor 4, nuclear factor-κB, cyclooxygenase-2, tumor necrosis factor-α) following miR-31-5p and SATB2 modulation in HCT116 cells. ^aP < 0.05 vs NC + vector, ^bP < 0.01 vs NC + vector, ^dP < 0.05 vs vector + mimic, ^eP < 0.01 vs vector + mimic, ^gP < 0.05 vs NC + OE-SATB2, ^hP < 0.01 vs NC + OE-SATB2 (n = 3); G: Western blots of mitochondrial apoptosis-related proteins (Bcl-2, Bax, cytochrome C, caspase-9, caspase-3) assessing SATB2-mediated reversal of miR-31-5p anti-apoptotic signaling in HCT116 cells. ^aP < 0.05 vs NC + vector, ^bP < 0.01 vs NC + vector, ^dP < 0.05 vs vector + mimic, ^eP < 0.01 vs vector + mimic, ^gP < 0.05 vs NC + OE-SATB2, ^hP < 0.01 vs NC + OE-SATB2 (n = 3). TLR-4: Toll-like receptor 4; NF-κB: Nuclear factor-κB; COX-2: Cyclooxygenase-2; TNF-α: Tumor necrosis factor-α; Cyt-C: Cytochrome C.

Figure 6 Resveratrol suppresses the development of azoxymethane/dextran sulfate sodium-induced colitis-associated colorectal cancer. A: Schematic overview of the azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated colorectal cancer (CAC) model in C57BL/6 mice; B: Dynamic changes in body weight following AOM/DSS treatment with or without resveratrol. ^bP < 0.01 vs control; C: Representative hematoxylin and eosin stains of colonic tissues showing morphological alterations (scale bar = 50 μm, 100 μm); D: Effects of Res on colon length in AOM/DSS-induced CAC mice. ^bP < 0.01 vs control, ^dP < 0.05 vs AOM/DSS; E: Effects of Res on spleen index (spleen weight/body weight) in AOM/DSS-induced CAC mice. ^bP < 0.01 vs control, ^dP < 0.05 vs AOM/DSS; F: Western blots of SATB2 protein expression in colonic tissues from CAC mice treated with Res. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^dP < 0.05 vs AOM/DSS; G: Western blots of mitochondrial apoptosis-related protein expression levels (Bax, Bcl-2, cytochrome C, caspase-9, and caspase-3) in colonic tissues. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^eP < 0.01 vs AOM/DSS; H: Terminal deoxynucleotidyl transferase dUTP Nick End Labeling staining showing apoptotic cells in colonic tissues from CAC mice (scale bar = 50 μm). ^aP < 0.05 vs control, ^dP < 0.05 vs AOM/DSS; I: Immunohistochemistry stains of SATB2, proliferation markers (Ki67 and β-catenin), and inflammatory signaling proteins (Toll-like receptor 4 and nuclear factor-κB) in CAC mouse colonic tissues (scale bar = 100 μm). ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^dP < 0.05 vs AOM/DSS, ^eP < 0.01 vs AOM/DSS; J: Hematoxylin and eosin staining of heart, liver, and kidney tissues showing no apparent toxicity of Res in CAC mice (scale bar = 50 μm, 100 μm). NS: Not significant; AOM: Azoxymethane; DSS: Dextran sulfate sodium; Res: Resveratrol; Cyt-C: Cytochrome C; TLR-4: Toll-like receptor 4; NF-κB: Nuclear factor-κB; IHC: Immunohistochemistry.

Figure 7 Resveratrol suppresses malignant phenotypes of HCT116 cells via the miR-31-5p/SATB2 axis. A: CCK-8 assay of HCT116 cell viability after 24 hours of exposure to Resveratrol (Res) (0, 2.5, 5, 10, 20, and 40 μg/mL). ^aP < 0.05 vs 0 μg/mL, ^bP < 0.01 vs 0 μg/mL (n = 8); B: Quantitative reverse transcription polymerase chain reaction analysis of miR-31-5p in HCT116 cells treated with 10 μg/mL Res for 0, 12, or 24 hours. ^bP < 0.01 vs 0 hours (n = 3); C: Quantitative reverse transcription polymerase chain reaction quantification of miR-31 expression in colonic tissues from colitis-associated colorectal cancer mice with Res intervention. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^eP < 0.01 vs azoxymethane/dextran sulfate sodium (n = 3); D: Western blots of SATB2 protein expression in HCT116 cells treated with Res (0, 2.5, 5, 10, 20, and 40 μg/mL). ^bP < 0.01 vs control (n = 3); E and F: Cell migration evaluated by scratch wound assays under miR-31-5p overexpression or inhibition with Res treatment (scale bar = 200 μm). ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^eP < 0.01 vs miR-31-5p mimic, ^gP < 0.05 vs miR-31-5p inhibitor (n = 3); G: Colony formation assay evaluating the impact of Res on miR-31-5p-modulated proliferative capacity. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^dP < 0.05 vs miR-31-5p mimic, ^gP < 0.05 vs miR-31-5p inhibitor (n = 3); H: Flow cytometric analysis of apoptosis in HCT116 with altered miR-31-5p expression with/without Res treatment. ^bP < 0.01 vs control, ^eP < 0.01 vs miR-31-5p mimic, ^hP < 0.01 vs miR-31-5p inhibitor (n = 3); I: Western blots of SATB2 and nuclear factor-κB expression and the Bcl-2/Bax ratio after miR-31-5p manipulation with/without Res treatment. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^dP < 0.05 vs miR-31-5p mimic, ^eP < 0.01 vs miR-31-5p mimic, ^hP < 0.01 vs miR-31-5p inhibitor (n = 3); J and K: Scratch wound assays with SATB2-silenced (si-SATB2) or SATB2-overexpressing (OE-SATB2) HCT116 cells treated with Res (scale bar = 200 μm). ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^eP < 0.01 vs si-SATB2, ^gP < 0.05 vs OE-SATB2 (n = 3); L and M: Colony formation assays with si-SATB2 or OE-SATB2 cells with Res treatment. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^eP < 0.01 vs si-SATB2, ^gP < 0.05 vs OE-SATB2 (n = 3); N and O: Western blots of apoptosis-related protein expression levels in si-SATB2 or OE-SATB2 cells following Res treatment. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^dP < 0.05 vs si-SATB2, ^eP < 0.01 vs si-SATB2, ^gP < 0.05 vs OE-SATB2, ^hP < 0.01 vs OE-SATB2 (n = 3). Res: Resveratrol; NS: Not significant; NF-κB: Nuclear factor-κB; AOM: Azoxymethane; DSS: Dextran sulfate sodium; Cyt-C: Cytochrome C.