MicroRNA-582-3p targeting ribonucleotide reductase regulatory subunit M2 inhibits the tumorigenesis of hepatocellular carcinoma by regulating the Wnt/β-catenin signaling pathway

Abstract

Hepatocellular carcinoma (HCC) is an important cause of death worldwide. MicroRNA (miRNA)-mediated gene silencing is involved in tumor biology. In this study, we aimed to elucidate the function and mechanism of action of miR-582-3p in HCC. We performed reverse transcription-quantitative polymerase chain reaction and western blotting to detect the expression levels of miR-582-3p, ribonucleotide reductase regulatory subunit M2 (RRM2), and markers of the Wnt/β-catenin signaling pathway (Wnt, Gsk-3β, β-catenin, and C-myc). The potential binding between miR-582-3p and RRM2 was confirmed using a dual-luciferase reporter assay. The proliferative and migratory capacities of the cells were evaluated using the cell counting kit-8 and wound-healing assays, respectively. Mouse models were used to validate the role of miR-582-3p in vivo. We observed the downregulation of miR-582-3p levels in HCC tumors and cell lines. Its upregulation in Huh7 and Hep 3B cells impaired their proliferation and migration, and the in vivo results showed suppressed tumor growth. Additionally, miR-582-3p upregulation also reduced the expression levels of Wnt, β-catenin, and C-myc, but enhanced the expression levels of glycogen synthase kinase-3β, both in vitro and in vivo. miR-582-3p targeted RRM2, and a negative correlation was observed in its expression patterns in HCC. Furthermore, RRM2 overexpression aggravated the proliferative and migratory capabilities of Hep3B and Huh7 cells and triggered Wnt/β-catenin signaling. However, miR-582-3p depleted RRM2 expression, thereby attenuating the oncogenic effects of RRM2. In conclusion, our results demonstrated that miR-582-3p binds to RRM2 to regulate the Wnt/β-catenin signaling pathway, thereby blocking the progression of HCC.

Keywords: Hepatocellular carcinoma; RRM2; miR-582-3p; wnt/β-catenin.

Graphical abstract

Figure 1.

MiR-582-3p upregulation suppressed the cancerous phenotypes of HCC cells and inactivated the Wnt/β-catenin pathway. (a) Relative expressions of miR-582-3p in normal tissues and tumors were checked via RT-qPCR, P < 0.0001. (b) Relative expression of miR-582-3p among the THLE2, Huh7, SNU-182, Hep 10 and Hep 3B cell lines was checked via RT-qPCR. (c) The overexpression efficiency of miR-582-3p mimic in Hep 3B and Huh7 cell lines as measured via RT-qPCR. (d) Effects of miR-582-3p mimic on cell proliferation, determined by CCK-8 assay. (e) MiR-582-3p mimic’s effect on cell migration was determined by wound healing assay. (f) Relative protein expression levels of Wnt, β-catenin, C-myc, GSK-3β, and p-GSK-3β in miR-582-3p mimic-transfected cells was quantified via western blot experiment. B: *P < 0.05 and **P < 0.001 vs. THLE2; C-F: *P < 0.05 and **P < 0.001 vs. mimic-NC.

Figure 2.

MiR-582-3p impeded the growth of tumors and inactivated the Wnt/β-catenin pathway in the animal models. (a) Images of the excised tumors from animal models. (b) Tumor volume in nude mice injected with Hep 3B cells overexpressing miR-582-3p or NC. (c) After 28 days, euthanasia was carried out on the mice before their tumors were surgically removed and weighed. (d) The protein levels of RRM2, Wnt, β-catenin, C-myc, GSK-3β, and p-GSK-3β in the excised tumors of both the groups were measured via western blotting. *P < 0.05 and **P < 0.001 vs. NC.

Figure 3.

RRM2 3ʹUTR and MiR-582-3p interacted. (a) The prospective binding sites between RRM2 3ʹUTR and miR-582-3p were predicted with the aid of starBase. (b) RRM2 and miR-582-3p binding was confirmed via dual-luciferase reporter experiment, **P < 0.001 vs. miR-NC. (c) Relative levels of RRM2 in tumors and normal tissues were assessed via RT-qPCR. (d) RRM2 expression in THLE2, Hep 3B and Huh7 cells was determined by RT-qPCR, **P < 0.001 vs. THLE2. (e) Pearson’s test uncovered an inverse association between the expressions of miR-582-3p and RRM2 in tumors.

Figure 4.

MiR-582-3p upregulation suppressed RRM2 to inhibit HCC cell malignant phenotypes and block the activity of the Wnt/β-catenin signaling. (a) In Hep 3B and Huh7 cells transfected with empty vector, mimic-NC, RRM2-OE, mimic, or OE+mimic, the relative RRM2 protein expression was determined via western blotting. (b-c) The viability (b) and migratory capacity (c) of the transfected Huh7 and Hep 3B cell lines were evaluated via CCK-8 and wound-healing assays, respectively. (d) The protein levels of Wnt, β-catenin, C-myc and p-Gsk-3β in the transfected cells were measured via western blotting. #P < 0.05 and ##P < 0.001 vs. mimic-NC; *P < 0.05 and **P < 0.001 vs. Empty vector; &P < 0.05 and &&P < 0.001 vs. OE+mimic.