MicroRNA-206 suppresses hypoxia-inducible factor-1α/PFKFB3-mediated glycolysis to inhibit recurrence and metastasis of hepatocellular carcinoma after incomplete radiofrequency ablation

Figure 1 Flowchart of the clinical study design. miR-206: MicroRNA-206; HIF-1α: Hypoxia-inducible factor-1α.

Figure 2 Changes in serum biomarker levels in patients undergoing complete or incomplete radiofrequency ablation. A: Serum hypoxia-inducible factor-1α levels measured preoperatively and 1 week after radiofrequency ablation (RFA) in the complete ablation group (n = 35) and incomplete ablation group (n = 10); B: Serum microRNA-206 levels in the same patient cohorts (n = 35 vs n = 10); C: Changes in serum glucose levels before and after RFA (n = 45); D: Changes in serum pyruvate levels before and after RFA (n = 45). Data are presented as mean ± SD. Student’s t-test was used for comparisons. ^aP < 0.05; ^bP < 0.01. RFA: Radiofrequency ablation; miR-206: MicroRNA-206; HIF-1α: Hypoxia-inducible factor-1α.

Figure 3 Functional changes in tumor-derived endothelial cells after thermal stimulation. A: Wound-healing migration assay at 0 h and 48 h under 37 °C or 41 °C (n = 3); B: Quantification of wound-closure rate (n = 3); C: Transwell migration assay (n = 3); D: Transwell invasion assay (n = 3); E: Apoptosis measured by TUNEL staining at 37 °C, 41 °C, 44 °C, and 47 °C (n = 3); F: Western blot analysis of hypoxia-inducible factor-1α, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3, and GAPDH under each temperature condition (n = 3); G: Tube-formation assay under 37 °C, 41 °C, 44 °C, and 47 °C (n = 3). Data are expressed as mean ± SD; one-way ANOVA was used for statistical analysis. ^aP < 0.05. Td-EC: Tumor-derived endothelial cells.

Figure 4 Effects of microRNA-206 overexpression and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 knockdown on endothelial function and glycolysis under thermal stimulation. A: Tube-formation assay under 37 °C and 41 °C in control, mimic NC, microRNA-206 (miR-206) mimics, shRNA NC, and sh-6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) groups (n = 3); B and C: TUNEL apoptosis staining and quantification under the same conditions (n = 3); D: Transwell migration assay results comparing miR-206 and sh-PFKFB3 effects (n = 3); E: Western blot analysis of hypoxia-inducible factor-1α, PFKFB3, and GAPDH under all conditions (n = 3). Data represent mean ± SD; one-way ANOVA was used. ^aP < 0.05. Td-EC: Tumor-derived endothelial cells; HIF-1α: Hypoxia-inducible factor-1α; PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3.

Figure 5 Angiogenesis, glycolytic signaling, and protein expression in rabbit VX2 liver tumors after complete or incomplete radiofrequency ablation. A: Immunofluorescence staining of α-SMA in sham, complete radiofrequency ablation (RFA), and incomplete RFA groups at 0 week, 1 week, and 3 weeks (n = 6 animals per group per timepoint); B: Quantification of α-SMA average optical density (AOD) (n = 6); C: Western blot analysis of hypoxia-inducible factor-1α (HIF-1α), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3, and GAPDH across treatment groups and timepoints (n = 6); D: Immunohistochemistry (IHC) of HIF-1α expression at each timepoint (n = 6); E: Quantification of HIF-1α IHC AOD (n = 6). Data are shown as mean ± SD; one-way ANOVA was used for comparisons. ^aP < 0.05. RFA: Radiofrequency ablation; HIF-1α: Hypoxia-inducible factor-1α; PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3; AOD: Average optical density.