Glaucocalyxin A Inhibits the Malignant Progression of Epithelial Ovarian Cancer by Affecting the MicroRNA-374b-5p/HMGB3/Wnt-β-Catenin Pathway Axis

Abstract

Objective: Glaucocalyxin A (GLA) is an ent-kaurene diterpenoid from Rabdosia japonica var possessing anti-tumor activity. This study aimed to investigate effects of GLA on epithelial ovarian cancer (EOC) and elucidate underlying mechanisms.

Methods: The expression of HMGB3 in EOC tissues was analyzed by GEPIA and immunohistochemistry. Cell proliferation was determined using CCK-8 and colony formation assays. Cell invasion, migration, and apoptosis were detected using Transwell, wound healing, and flow cytometry assays, respectively. Interactions between HMGB3 and miRNAs were predicted using ENCORI and validated using a dual-luciferase assay. mRNA expression levels of HMGB3 and miRNAs were measured using qPCR. Protein expression levels of HMGB3, E-cadherin, N-cadherin, Wnt3a,β-catenin, Bcl-2, and Bax were measured by western blotting. A tumor xenograft model was established to validate the efficacy and mechanism of GLA in vivo.

Results: HMGB3 was upregulated in EOC tissues and cells. GLA dose-dependently inhibited EOC cell proliferation and epithelial-mesenchymal transition (EMT). HMGB3 overexpression promoted proliferation, invasion, migration, and EMT, and suppressed the apoptosis of EOC cells. In addition, miR-374b-5p was targeted by HMGB3, and its overexpression hindered malignant characteristics of EOC cells. HMGB3 overexpression weakened antitumor effects of GLA and miR-374b-5p in EOC cells. Moreover, the Wnt-β-catenin pathway was inhibited by the GLA-mediated miR-374b-5p/HMGB3 axis. In vivo experiments showed that GLA inhibited EOC tumor growth, meanwhile, upregulated the miR-374b-5p level and downregulated the expression of HMGB3, Wnt3a, and β-catenin in tumor tissues.

Conclusions: GLA suppressed the malignant progression of EOC by regulating the miR-374b-5p/HMGB3/Wnt-β-catenin pathway axis.

Keywords: Wnt-β-catenin pathway; epithelial ovarian cancer; glaucocalyxin A; high-mobility group box 3; microRNA-374b-5p.

Figure 1

HMGB3 was highly expressed in epithelial ovarian cancer (EOC) tissues and cells. (A) The HMGB3 expression profile was analyzed in EOC tissues according to the data on the gene expression profiling interactive analysis (GEPIA, http://gepia.cancer-pku.cn/). ^* P < 0.05. (B) HMGB3 expression in human EOC tumor tissues and normal tissues was detected by immunohistochemistry (100× magnification). (C) The mRNA expression of HMGB3 in EOC cell lines (SKOV3 and OVCAR3) and the normal human ovarian epithelial cell line (IOSE80) was measured by qPCR. (D) Relative protein expression of HMGB3 in EOC cell lines (SKOV3 and OVCAR3) and the normal human ovarian epithelial cell line (IOSE80) was determined by western blotting. ^* P < 0.05 and ^** P < 0.01 vs. IOSE80.

Figure 2

Glaucocalyxin A (GLA) dose-dependently inhibited proliferation, epithelial-mesenchymal transition (EMT), and HMGB3 expression in EOC cells. (A, B) The proliferation of IOSE80 and SKOV3 cells was measured by CCK-8 assay. (C) The IC50 value of GLA was determined by a proliferation curve at 48 h post-treatment. (D, E) The protein levels of HMGB3 and EMT-related biomarkers (E-cadherin and N-cadherin) in SKOV cells were detected by western blotting. EOC cells were treated with 1, 2, 4, 8, and 16 μmol/L GLA, respectively. *P <0.05 and **P <0.01 vs. control..

Figure 3

GLA inhibited the malignant characteristics of EOC cells by regulating HMGB3. (A) The relative mRNA expression of HMGB3 in SKOV3 cells was measured by qPCR. (B) The viability of SKOV3 cells was detected by CCK-8 assay. (C) The proliferation of SKOV3 cells was detected by colony formation assay. (D) The apoptosis of SKOV3 cells was detected by flow cytometry. (E) The migration of SKOV3 cells was detected by wound healing assay. (F) The invasion of SKOV3 cells was detected by the Transwell assay. Scale bar = 50 µm. SKOV3 cells were treated with GLA, sh-HMGB3/sh-NC, and/or lenti-HMGB3/lenti-NC. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 4

GLA inhibited the expression of EMT and Wnt-β-catenin pathway-related proteins by regulating HMGB3. The relative protein expression of E-cadherin, N-cadherin, HMGB3, Wnt3a, and β-catenin in SKOV3 cells was measured by western blotting. SKOV3 cells were treated with GLA, sh-HMGB3/sh-NC, and/or lenti-HMGB3/lenti-NC. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 5

MiR-374b-5p was predicted to directly target HMGB3. (A) The relative expression of miR-374b-5p, miR-429, and miR-214-3p in EOC cell lines (SKOV3 and OVCAR3) and those of normal IOSE80 cells was detected by qPCR. ^* P < 0.05 vs. IOSE80. (B) The binding site of has-miR-374b-5p and HMGB3. (C) The interaction between miR-374b-5p and HMGB3 was validated through dual-luciferase reporter assay. ^** P < 0.01.

Figure 6

GLA inhibited the malignant characteristics of EOC cells by affecting miR-374b-5p/HMGB3 axis. (A) The relative mRNA expression of miR-374b-5p and HMGB3 in SKOV3 cells was measured by qPCR. (B) The viability of SKOV3 cells was detected by CCK-8 assay. (C) The proliferation of SKOV3 cells was detected by colony formation assay. (D) The apoptosis of SKOV3 cells was detected by flow cytometry. (E) The migration of SKOV3 cells was detected by wound healing assay. (F) The invasion of SKOV3 cells was detected by the Transwell assay. Scale bar = 50 µm. SKOV3 cells were treated with GLA, miR-374b-5p mimics/NC mimics, and/or lenti-HMGB3/lenti-NC. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 7

GLA inhibited the expression of EMT and Wnt-β-catenin pathway-related proteins via regulating miR-374b-5p/HMGB3 axis. The relative protein expression of E-cadherin, N-cadherin, HMGB3, Wnt3a, and β-catenin in SKOV3 cells in SKOV3 cells was detected by western blotting. SKOV3 cells were treated with GLA, miR-374b-5p mimics/NC mimics, and/or lenti-HMGB3/lenti-NC. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 8

GLA restrained the EOC tumor growth via affecting miR-374b-5p/HMGB3/Wnt-β-catenin pathway axis. BALB/c nude mice were subcutaneously injected SKOV3 cells and treated with low- or high-dose GLA. (A) Tumor volume and weight were recorded. (B) The expression of Ki-67 (a cell proliferation marker) and HMGB3 in tumor tissues was identified by immunohistochemistry. Scale bar = 20 µm. (C) The expression of miR-374b-5p in tumor tissues. (D) The protein levels of HMGB3, Wnt3a, and β-catenin in tumor tissues. *P <0.05 and **P <0.01 vs. control; ##P <0.01 vs. the low- GLA.