Tumor suppressor role of sFRP‑4 in hepatocellular carcinoma via the Wnt/β‑catenin signaling pathway

Abstract

Hepatocellular carcinoma (HCC) is a malignant tumor located in the liver. Secreted frizzled‑related protein 4 (sFRP‑4) is associated with cancer occurrence, but the relationship between sFRP‑4 and HCC is not completely understood. The present study aimed to investigate the role and mechanism underlying sFRP‑4 in HCC. sFRP‑4 mRNA expression levels were determined via reverse transcription‑quantitative PCR and immunohistochemistry. The Cell Counting Kit‑8 assay was performed to evaluate HCCLM3 and Huh7 cell viability. Moreover, HCCLM3 and Huh7 cell proliferation were assessed using the BrdU ELISA assay kit, and cell apoptosis was measured via flow cytometry. Western blotting was conducted to measure β‑catenin and GSK‑3β protein expression levels. The results demonstrated that sFRP‑4 expression was significantly downregulated in HCC tissues and cells compared with adjacent healthy tissues and MIHA cells, respectively. Moreover, the results indicated that compared with the control group, sFRP‑4 overexpression inhibited HCC cell viability and proliferation, and accelerated HCC cell apoptosis. Furthermore, the results suggested that sFRP‑4 inhibited the Wnt/β‑catenin signaling pathway by upregulating GSK‑3β expression and downregulating β‑catenin expression, thus restraining the malignant behavior of HCC cells. In conclusion, the present study indicated that sFRP‑4 served a tumor suppressor role in HCC cells by restraining the Wnt/β‑catenin signaling pathway.

Keywords: secreted frizzled‑related protein 4; hepatocellular carcinoma; Wnt/β‑catenin.

Figure 1.

sFRP-4 is downregulated in HCC tissues and cells. (A) sFRP-4 mRNA expression levels in HCC tissues and adjacent healthy tissues were measured via RT-qPCR (n=47). Data were analyzed using the paired Student's t test. (B) Immunohistochemistry was performed to evaluate sFRP-4 expression in 8 paired HCC and adjacent healthy tissues. Images a01-a08 represent HCC tissues and images b01-b08 represent the corresponding adjacent healthy tissues (magnification, ×200). (C) sFRP-4 mRNA expression levels in HCC cell lines (HCCLM3, Hep3B and Huh7) and a normal human liver cell line (MIHA) were detected via RT-qPCR. Data were analyzed using one-way ANOVA followed by Bonferroni's post hoc test. Data are presented as the mean ± SD from three independent experiments. *P<0.05 and **P<0.001 vs. MIHA. sFRP-4, secreted frizzled-related protein 4; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative PCR.

Figure 2.

sFRP-4 inhibits HCC cell viability and proliferation, and promotes HCC cell apoptosis. (A) Transfection efficiency of sFRP-4-OE and si-sFRP-4 in HCCLM3 and Huh7 cells. (B) Effect of sFRP-4 on HCCLM3 and Huh7 cell viability, as evaluated by performing CCK-8 assays. (C) Effect of sFRP-4 on HCCLM3 and Huh7 cell proliferation, as evaluated by performing BrdU ELISA assays. Effect of sFRP-4 on HCCLM3 and Huh7 cell apoptosis was (D) evaluated via flow cytometry and (E) quantified. Data were analyzed using one-way ANOVA followed by Bonferroni's post hoc test. Data are presented as the mean ± SD from three independent experiments. *P<0.05 and **P<0.001 vs. CON. sFRP-4, secreted frizzled-related protein 4; HCC, hepatocellular carcinoma; OE, overexpression; si, small interfering RNA; CCK-8, Cell Counting Kit-8; CON, blank control; NC, negative control (empty vectors and si-NC); OD, optical density.

Figure 3.

sFRP-4 inhibits the Wnt/β-catenin signaling pathway in HCC. GSK-3β and β-catenin protein expression levels in HCCLM3 and Huh7 cells transfected with sFRP-4-OE, si-sFRP-4 or NC were (A) determined via western blotting and (B) semi-quantified. (C) GSK-3β, β-catenin and sFRP-4 protein expression levels in HCCLM3 and Huh7 cells following treatment with IM-12 or DMSO for 36 h were determined via western blotting and semi-quantified. Data were analyzed using one-way ANOVA followed by Bonferroni's post hoc test. Data are presented as the mean ± SD from three independent experiments. **P<0.001 vs. CON or DMSO. sFRP-4, secreted frizzled-related protein 4; HCC, hepatocellular carcinoma; OE, overexpression; si, small interfering RNA; NC, negative control (empty vectors and si-NC); CON, blank control.

Figure 4.

sFRP-4 inhibits HCC cell viability and proliferation, and promotes HCC cell apoptosis via the Wnt/β-catenin signaling pathway. (A) HCCLM3 and Huh7 cell viability following transfection with sFRP-4-OE and treatment with IM-12 for 36 h was determined by performing CCK-8 assays. (B) HCCLM3 and Huh7 cell proliferation following transfection with sFRP-4-OE and treatment with IM-12 for 36 h was determined by performing BrdU ELISA assays. HCCLM3 and Huh7 cell apoptosis following transfection with sFRP-4-OE and treatment with IM-12 for 36 h was (C) determined via flow cytometry and (D) quantified. Data were analyzed using one-way ANOVA followed by Bonferroni's post hoc test. Data are presented as the mean ± SD from three independent experiments. *P<0.05 and **P<0.001 vs. CON; ^##P<0.001 vs. sFRP-4-OE. sFRP-4, secreted frizzled-related protein 4; HCC, hepatocellular carcinoma; OE, overexpression; CCK-8, Cell Counting Kit-8; CON, blank control; NC, negative control; OD, optical density; IM-12, a selective GSK-3β inhibitor.