Fig 2: MMP16 is the direct target of miR-193a-5p. A, Bioinformatics analysis was performed with starBase, TargetScan, and miRDB to predict the candidate mRNAs, which could be targeted by miR-193a-5p. B, Bioinformatics analysis predicted that the sequence of MMP16 3UTR matched the sequences of miR-193a-5p, and MMP16-WT (wild-type) and MMP16-MUT (mutant) luciferase reporter gene vectors were constructed. C, The miR-193a-5p mimics (or miR control) were co-transfected with MMP16-WT (or MMP16-MUT) into HEK293T cells, and the luciferase activity of the cells in each group was measured. D, Huh-7 and HepG2 cells were transfected with anti-miR-193a-5p and negative control (NC), and the expression level of miR-193a-5p was detected by qRT-PCR. E, qRT-PCR was used to detect the expression of MMP16 mRNA in Huh-7 and HepG2 cells after circ_0001806 knockdown. F, qRT-PCR was used to detect the expression of MMP16 mRNA in Huh-7 and HepG2 cells transfected with anti-miR-193a-5p. G, Western blot was used to detect the expression of MMP16 protein in Huh-7 and HepG2 cells after circ_0001806 knockdown (G) and in Huh-7 and HepG2 cells transfected with anti-miR-193a-5p (H). Data are reported as means±SD. **P<0.01, ***P<0.001 (t-test).

Fig 3: NLRP3 overexpression abrogated the anti-tumor effects of eriodictyol in vivo. (A) Images of subcutaneous tumors of control, eriodictyol, and eriodictyol + NLRP3 group mice (n = 5/per group). (B) The growth curve of xenograft tumor in the control, eriodictyol, and eriodictyol + NLRP3 groups. (C) Tumor weight in the control, eriodictyol, and eriodictyol + NLRP3 groups. (D) The growth curve of body weight of mice in the control, eriodictyol, and eriodictyol + NLRP3 groups. (E, F) Pathological damage of the tumor and liver was detected by H&E staining. (G) Immunohistochemical staining of NLRP3, VEGFA, MMP3, and MMP16 in tumor tissues. (H) Positive staining rates of NLRP3, VEGFA, MMP3, and MMP16. (I) The levels of AST and ALT were determined by ELISA. (**p < 0.01 vs control group, ##p < 0.01 vs eriodictyol group).

Fig 4: Lactate in the TME as a possible signaling molecule in support of PEPT1 function.Real-time PCR showing relative mRNA expression of MMP9 (A), MMP13 (B), MMP16 (C), and DPPIV (D) in control and lactate-treated SU.86.86 cells. Western blotting showing protein expression of MMP9, MMP13, MMP16, and DPPIV from total cell lysates of cultured Capan-1 and SU.86.86 vs. the tumor lysates from Capan-1 and SU.86.86 xenografted tumors. Beta-actin was used as a loading control (E). Real-time PCR showing relative PEPT1 mRNA expression in amino acid- and serum-starved Capan-1 and SU.86.86 cells restimulated with Phe (F and G) and Gly-Pro (H and I). PEPT1-mediated [3H]Gly-Sar uptake in amino acid- and serum-starved Capan-1 and SU.86.86 cells restimulated with Phe (J and N), Lys (K and O), Gly-Sar (L and P), and Gly-Phe (M and Q). Data are given as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 5: Circ_0001806 enhances the multiplication and invasion of hepatocellular carcinoma (HCC) cells through miR-193a-5p/MMP16 axis. Huh-7 and HepG2 cells were transfected with si-NC + anti-miR-Con, si-circ_0001806#1 + anti-miR-Con, or si-circ_0001806#1 + anti-miR-193a-5p. A, qRT-PCR was used to detect the expression of miR-193a-5p in Huh-7 and HepG2 cells. B, qRT-PCR was used to detect the expression of MMP16 mRNA in Huh-7 and HepG2 cells. C, Western blot was used to detect the expression of MMP16, MMP2, and MMP9 protein in Huh-7 and HepG2 cells. D, CCK-8 method was used to detect the multiplication of Huh-7 and HepG2 cells after transfection. The transwell experiment was employed to detect the migration and invasion of Huh-7 cells (E) and HepG2 cells (F) after transfection (scale bar 20 μm). Data are reported as means±SD. *P<0.05, **P<0.01, and ***P<0.001 (ANOVA).