Effects and related mechanisms of neutrophil extracellular traps on proliferation and migration abilities of breast cancer
10.3760/cma.j.cn115355-20240606-00282
- VernacularTitle:中性粒细胞胞外诱捕网对乳腺癌细胞增殖和迁移能力的影响及相关机制
- Author:
Ying ZHOU
1
;
Huitao XU
1
;
Huanhuan ZHANG
1
;
Chu ZHANG
1
;
Shaolin ZHAO
1
;
Jin YANG
1
Author Information
1. 连云港市第一人民医院检验科，连云港 222002
- Publication Type:Journal Article
- Keywords:
Breast neoplasma;
Neutrophil extracellular traps;
Frizzled homolog 10;
Cell proliferation;
Cell migration
- From:
Cancer Research and Clinic
2025;37(2):93-100
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effects of neutrophil extracellular traps (NET) on the proliferation and migration of breast cancer cells and the related mechanisms.Methods:The peripheral blood neutrophils were separated using density gradient centrifugation, 100 nmol/L phorbol 12-myristate 13-acetate (PMA) was used to stimulate the formation of NET. Breast cancer cell line MDA-MB-231 was selected and treated with NET (NET group) or deoxyribonucleaseⅠ (DNase Ⅰ) digested NET (NET+DNase Ⅰ group),normal cultured MDA-MB-231 cells were used as the control group. In MDA-MB-231 cells of each group; cell proliferation ability was detected using CCK-8 assay; cell migration was detected by cell scratch assay and Transwell assay; real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expressions of frizzled homolog 10 (FZD10), cyclin D1, E-cadherin and Vimentin immunofluorescence assay was performed to detect the formation of NET and the expressions of E-cadherin and Vimentin; Western blotting was used to detected the expression of FZD10-Wnt-β-catenin signaling pathway-related proteins.Results:Neutrophils were stimulated with PMA for 4 hours, and immunofluorescence assay results showed that NET exhibited fibrous reticular structure with high expression of citrullinated histone (citH3) and myeloperoxidase (MPO). The CCK-8 assay results showed that the absorbance values of cells in the control group, NET group and NET+DNase Ⅰ group were 1.25±0.06, 2.14±0.15 and 1.47±0.11, respectively, and the difference was statistically significant ( F = 80.60, P < 0.001). The results of scratch assay displayed that the percentage of wound closure in the control group, NET group and NET+DNase Ⅰ group were (36.2±1.3)%, (67.2±2.0)% and (46.3±3.2)%, respectively, and the difference was statistically significant ( F = 140.50, P < 0.001). Transwell assay indicated that the number of migrating cells in the control group, NET group and NET+DNase Ⅰ group were 317±18, 512±23 and 356±23, respectively, and the difference was statistically significant ( F = 75.39, P < 0.001). qRT-PCR assay revealed that the relative expressions of FZD10, cyclin D1 and Vimentin mRNA in the NET group were higher than those in the control group, while the relative expression of E-cadherin mRNA was lower than that in the control group, and the differences were statistically significant (all P < 0.001); the relative expressions of FZD10, cyclin D1 and Vimentin mRNA in the NET+DNase Ⅰ group were lower than those in the NET group, while the relative expression of E-cadherin mRNA was higher than that in the NET group, and the differences were statistically significant (all P < 0.001). Immunofluorescence assay indicated that the number of cells expressing Vimentin in the control group, NET group and NET+DNase Ⅰ group were 35±6, 86±13 and 51±6, respectively, and the difference was statistically significant ( F = 24.65, P = 0.001); the number of cells expressing E-cadherin were 31±4, 11±3 and 24±2, respectively, and the difference was statistically significant ( F = 38.36, P < 0.001). Western blotting results demonstrated that the relative expressions of FZD10, β-catenin and Vimentin proteins in the NET group were higher than those in the control group, while the relative expression of E-cadherin protein was lower than that in the control group; the relative expressions of FZD10, β-catenin and Vimentin proteins in the NET+DNase Ⅰ group were lower than those in the NET group, while the relative expression of E-cadherin protein was higher than that in the NET group. Conclusions:NET may promote the proliferation and migration of breast cancer cells by upregulating the expression of FZD10 and subsequently activating the Wnt-β-catenin signal pathway.
