ZWINT down-regulated by miR-495-3p inhibited lung metastasis of breast cancer by blocking p38 MAPK signaling pathway activation

Abstract

Breast cancer metastasis is the primary cause of patient mortality, yet effective therapeutic targets remain limited. Building on our prior identification of ZWINT as a prognostic marker linked to metastasis, this study defines its critical functional role and regulatory mechanism. Multi-omics analysis revealed a strong association between ZWINT expression and metastatic processes across breast cancer subtypes. Functionally, ZWINT knockdown significantly inhibited breast cancer cell migration and invasion in vitro and dramatically reduced lung metastasis in vivo. Mechanistically, we discovered that miR-495-3p directly targets and suppresses ZWINT expression, and this miR-495-3p/ZWINT axis acts through inhibiting p38 MAPK pathway activation to suppress metastatic progression in vitro. Our findings demonstrate that ZWINT drives breast cancer metastasis and is negatively regulated by miR-495-3p. The newly identified miR-495-3p/ZWINT/p38 MAPK axis may provide a promising therapeutic target for suppressing breast cancer progression.

Keywords: Breast cancer; Lung metastasis; MiR-495-3p; P38 MAPK pathway; ZWINT.

Fig. 1

ZWINT knockdown inhibits migration and invasion of breast cancer cells in vitro. A RT-PCR and western blot analysis of ZWINT mRNA and protein levels in MCF-10A, MCF-7, and MDA-MB-231 cells. B, D Wound-healing assay showed the migration rates of the control and ZWINT-silenced MCF-7/MDA-MB-231 cells. Bar = 100 μm. C, E Transwell invasion assay showing decreased invasion rates in ZWINT-silenced MCF-7 and MDA-MB-231 cells compared to controls. Bar = 100 μm. N = 3, *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 2

ZWINT knockdown inhibits breast cancer cell migration and invasion by modulating the p38 MAPK pathway. A Western blot analysis of p38 MAPK (p38) and phospho-p38 MAPK (p-p38) protein levels in control and ZWINT-silenced MCF-7/MDA-MB-231 cells. B Wound-healing assay was used to detect the effect of Anisomycin on the migration of the control and ZWINT-silenced MCF-7/MDA-MB-231 cells. Bar = 100 μm. C Transwell chamber invasion assay was used to detect the effect of Anisomycin on the invasion of the control and ZWINT-silenced MCF-7/MDA-MB-231cells. Bar = 100 μm. Anisomycin (2 μg/L), an agonist of p38 MAPK signaling pathway. Bar = 100 μm. N = 3, *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 3

ZWINT knockdown suppresses breast cancer lung metastasis and p38 MAPK pathway in vivo. A Representative morphology of lung tissue. B Quantification of pulmonary metastatic nodules. C HE staining of lung tissue, demonstrating metastatic lesions. Bar = 100 μm, and the black arrow indicates the nodule. D The impact of ZWINT knockdown on the protein expression levels of p38 MAPK (p38), phospho-p38 MAPK (p-p38), and the p-p38/p38 ratio in lung metastatic tissues from nude mice was assessed using western blot analysis. N = 3–6, *P < 0.05 and ***P < 0.001

Fig. 4

miR-495-3p directly targets ZWINT to inhibit its expression. A Three miRNAs (miR-495-3p, miR-124-3p, and miR-506-3p) were predicted to have potential binding sites with ZWINT based on miRanda, miRap, mircoT, and PITA software. B Kaplan–Meier plotter showed that breast cancer patients with high expression of miR-495-3p have a high survival rate. C miR-495-3p levels in breast cancer cells transfected with mimics/inhibitors or negative controls were quantified by RT-qPCR. D Wound-healing assay assessed migration of breast cancer cells transfected with miR-495-3p mimics/inhibitor and their negative control. Bar = 100 μm. E Transwell invasion assay evaluated invasion of breast cancer cells transfected with miR-495-3p mimics/inhibitor and their negative control. Bar = 100 μm. F RT-qPCR analysis of ZWINT mRNA levels in breast cancer cells transfected with miR-495-3p mimics/inhibitor and their negative control. G Western blot analysis of ZWINT protein levels in breast cancer cells transfected with miR-495-3p mimics/inhibitor and their negative control. H Luciferase reporter assay was used to analyze the interaction between miR-495-3p and ZWINT. NC, negative control. miR miR-495-3p, WT wild type, Mut mutant type. N = 3, ^N.S.P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 5

Overexpression of miR-495-3p inhibits breast cancer cell migration and invasion and suppresses the p38 MAPK pathway by downregulating ZWINT. A RT-PCR and western blot assessed ZWINT mRNA and protein levels in breast cancer cells transfected with miR-495-3p alone or combined with ZWINT. B, C A wound-healing assay assessed the migration of breast cancer cells transfected with miR-495-3p alone or combined with ZWINT. Bar = 100 μm. D, E Transwell invasion assay was used to detect the invasion ability of breast cancer cells transfected with miR-495-3p alone or in combination with ZWINT. Bar = 100 μm. F Western blot analysis of p38 MAPK (p38), phospho-p38 MAPK (p-p38), and the p-p38/p38 ratio was performed in breast cancer cells transfected with miR-495-3p alone or co-transfected with ZWINT. NC, negative control. miR miR-495-3p. N = 3, *P < 0.05, **P < 0.01, and ***P < 0.001