Exosome-derived miR-23a-5p inhibits HCC proliferation and angiogenesis by regulating PRDX2 expression: MiR-23a-5p/PRDX2 axis in HCC progression

Abstract

microRNAs (miRNAs) are closely related to the progression of hepatocellular carcinoma (HCC). Cancer-derived exosomes play an essential role in the establishment of the HCC microenvironment. However, the possible effects and underlying mechanisms of exosome (exo) microRNA-23a-5p (miR-23a-5p) in the progression of HCC remain unknown. In this study, we aimed to determine the role and specific molecular mechanism of exo miR-23a-5p in regulating HCC progression and to investigate whether exo miR-23a-5p levels can serve as an indicator of the prognosis of transarterial chemoembolization in patients with HCC. Our findings illustrated that miR-23a-5p was downregulated in exosomes separated from the serum of HCC patients and that miR-23a-5p carried by exosomes inhibited HCC cell proliferation and angiogenesis. Mechanistically, miR-23a-5p negatively targeted peroxiredoxin-2 (PRDX2). Functionally, PRDX2 overexpression relieved exosome-induced inhibition of HCC cell proliferation and angiogenesis by promoting vascular endothelial growth factor (VEGF) expression. In conclusion, Exo miR-23a-5p inhibited HCC proliferation and angiogenesis by regulating PRDX2 expression. Our results revealed the role and specific molecular mechanism of exo miR-23a-5p in regulating HCC progression.

Keywords: Angiogenesis; Exosomes; Hepatocellular carcinoma; PRDX2; miR-23a-5p.

Fig. 1

miR-23a-5p was downregulated in exosomes separated from the serum of HCC patients. Exosomes were separated from the serum of HCC patients after 3 days of TACE. A, Exosome features were observed by TEM. B, Exosome diameter was observed by DLS. C, The expression of CD81, TSG101, CD63 and Calnexin in exosomes was evaluated by Western blotting. D, The levels of miR-23a-5p, miR-155-3p, miR-135b, miR-130b-3p and miR-210-3p in the serum of HCC patients 3 days after TACE were measured by qRT‒PCR. E, miR-23a-5p levels in HCC cell lines, including HepG2, Hep3B, SKHep-1 and Huh-7, were measured by qRT‒PCR, and LX2 cells served as the negative control. **P < 0.01, ***P < 0.001.

Fig. 2

MiR-23a-5p carried by exosomes inhibited HCC cell proliferation and angiogenesis. HepG2 and SKHep-1 cells were randomly divided into four groups. In the control group, the cells were cultured normally. In the Exo group, cells were cocultured with exosomes separated from the serum of HCC patients after 3 days of TACE. In the Exos + inhibitor NC group, cells were cocultured with exosomes separated from the serum of HCC patients after 3 days of TACE and transfected with inhibitor NC, which served as the negative control for the miR-23a-5p inhibitor. In the Exos + miR-23a-5p inhibitor group, cells were cocultured with exosomes separated from the serum of HCC patients after 3 days of TACE and transfected with miR-23a-5p inhibitor for miR-23a-5p inhibition. A, qRT‒PCR measured miR-23a-5p levels in HepG2 and SKHep-1 cells. B, MTT assay detected HepG2 and SKHep-1 cell viability. C, Colony formation assay detected HepG2 and SKHep-1 cell proliferation. D, Tube formation assay detected tubule formation ability. E, ELISA measured the levels of MDA and SOD. F, DCFH-DA staining measured the level of ROS. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

PRDX2 knockdown promoted oxidative stress and inhibited HCC cell proliferation and angiogenesis, and miR-23a-5p negatively targeted PRDX2. A-B, Bioinformatics software TargetScan analysis showed that there were potential targets between PRDX2 and miR-23a-5p. C, A dual-luciferase reporter gene assay verified the targeting relationship between PRDX2 and miR-23a-5p. D, RIP assay further confirmed the targeting relationship between PRDX2 and miR-23a-5p. E, qRT‒PCR was used to measure PRDX2 levels in the serum of HCC patients 3 days after TACE. F, qRT‒PCR measured PRDX2 levels in HepG2 and SKHep-1 cells after transfection with miR-23a-5p inhibitor. G, qRT‒PCR was used to measure PRDX2 levels in HepG2 and SKHep-1 cells after transfection with miR-23a-5p mimics. H, Western blot analysis of PRDX2 levels in HepG2 and SKHep-1 cells after transfection with miR-23a-5p mimics. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4

PRDX2 overexpression relieved exosome-induced inhibition of HCC cell proliferation and angiogenesis by promoting VEGF expression. HepG2 and SKHep-1 cells were randomly divided into four groups. In the control group, the cells were cultured normally. In the Exo group, cells were cocultured with exosomes separated from the serum of HCC patients after 3 days of TACE. In the Exo + oe-NC group, cells were cocultured with exosomes separated from the serum of HCC patients after 3 days of TACE and transfected with oe-NC, which served as the negative control for oe-PRDX2. In the Exos + oe-PRDX2 group, cells were cocultured with exosomes separated from the serum of HCC patients after 3 days of TACE and transfected with oe-PRDX2 for PRDX2 overexpression. A, qRT‒PCR was used to measure PRDX2 mRNA levels. B, Western blot analysis of VEGF protein levels. C, MTT assay detected HepG2 and SKHep-1 cell viability. D, Colony formation assay detected HepG2 and SKHep-1 cell proliferation. E, Tube formation assay detected tubule formation ability. F, ELISA measure the levels of MDA and SOD. G, DCFH-DA staining measured the level of ROS. *P < 0.05, **P < 0.01, ***P < 0.001.