Effects of breast cancer mesenchymal stem cells on proliferation and migration of breast cancer MCF-7 cells via IL-6-STAT3 signaling pathway
10.3760/cma.j.cn115355-20230417-00191
- VernacularTitle:乳腺癌间质干细胞通过IL-6-STAT3信号通路影响乳腺癌MCF-7细胞增殖和迁移
- Author:
Ying ZHOU
1
;
Huitao XU
;
Huanhuan ZHANG
;
Chu ZHANG
;
Ping ZHENG
;
Jin YANG
Author Information
1. 连云港市第一人民医院检验科，连云港 222002
- Keywords:
Breast neoplasma;
Mesenchymal stem cells;
STAT3 transcription factor;
Interleukin-6;
Cell proliferation;
Cell migration;
Epithelial-mesenchymal transition
- From:
Cancer Research and Clinic
2023;35(11):801-807
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effects of breast cancer mesenchymal stem cells (BC-MSC) on the proliferation and migration of breast cancer MCF-7 cells and the related mechanisms.Methods:The resected cancer tissues and paracancerous tissues were taken from breast cancer patients after surgery, and the bone marrow samples of healthy people were selected. BC-MSC, breast cancer paracancerous mesenchymal stem cells (BCN-MSC) and bone marrow mesenchymal stem cells (BM-MSC) of healthy people were isolated and cultured by tissue adhesion method, and their differentiation ability was induced by the addition of osteogenic and lipogenic induction, and their surface markers were detected by flow cytometry. The supernatants of BC-MSC, BCN-MSC and BM-MSC of healthy people cultured for 48 h were collected and used for the culture of MCF-7 cells as BC-MSC group, BCN-MSC group and BM-MSC group, respectively, and the control group was the conventional cultured MCF-7 cells. The proliferation ability of MCF-7 cells in each group was detected by methyl thiazol tetrazolium (MTT) assay, the clone formation ability of MCF-7 cells was detected by plate cloning assay, the migration ability of MCF-7 cells was detected by Transwell assay, and the mRNA relative expressions of interleukin (IL)-6 and epithelial mesenchymal transition (EMT)-related genes (E-cadherin, vimentin, snail) were detected by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) in MCF-7 cells. Western blotting was used to detect expressions of p-STAT3, E-cadherin, vimentin and snail proteins in MCF-7 cells. Luminex liquid microarray technology was used to detect cytokine levels in culture supernatants of different mesenchymal stem cells (MSC). IL-6 neutralizing antibody was added into the supernatant of BC-MSC, MCF-7 cells were cultured with the supernatant (BC-MSC+IL-6 neutralizing antibody group), and then the proliferation and migration abilities of MCF-7 cells were tested, as well as the expression changes of related genes and proteins.Results:BC-MSC, BCN-MSC and BM-MSC were successfully isolated; BC-MSC had positive expressions of CD29, CD44 and CD90 and negative expressions of CD14, CD34 and CD45, which were in line with the characteristics of MSC. MTT assay showed that the absorbance values of MCF-7 cells cultured for 48 h in the control group, BC-MSC group, BCN-MSC group and BM-MSC group were 0.31±0.02, 0.54±0.03, 0.43±0.02 and 0.42±0.02, respectively, and the difference was statistically significant ( F = 56.52, P < 0.05); the results of plate cloning experiments showed that the number of clones in each petri dish of the four groups were 180±9, 439±17, 319±16 and 306±19, respectively, and the difference was statistically significant ( F = 222.70, P < 0.05); Transwell assay showed that the numbers of membrane-penetrating cells in the four groups were 6.5±1.0, 23.2±2.4, 16.0±1.3 and 14.8±2.0, respectively, with the statistically significant difference ( F = 49.44, P < 0.05); qRT-PCR assay showed that the relative expressions of IL-6 mRNA in the control group, BC-MSC group, BCN-MSC group and BM-MSC group were 1.07±0.11, 13.79±3.80, 6.68±1.66 and 6.12±1.52, respectively, and the difference was statistically significant ( F = 107.60, P < 0.05), and the relative expression of E-cadherin mRNA in MCF-7 cells of BC-MSC, BCN-MSC and BM-MSC groups was lower than that of the control group, while the relative expressions of vimentin and snail mRNA were higher than those of the control group, and the differences were statistically significant (all P < 0.05). Western blotting assay showed that the relative expression of E-cadherin mRNA in MCF-7 cells of BC-MSC, BCN-MSC and BM-MSC groups was lower than that of the control group. Western blotting showed that the level of E-cadherin protein in BC-MSC, BCN-MSC and BM-MSC groups was lower than that in the control group, and the levels of vimentin and snail proteins were higher than those in the control group; Luminex liquid microarray technology showed that the content of IL-6 cytokine in the supernatants of BC-MSC, BCN-MSC and BM -MSC cultures were higher, and the relative expressions were 1.75±0.21, 1.00±0.10 and 0.96±0.08, respectively, and the difference was statistically significant ( F = 43.22, P < 0.05). The results of MTT assay showed that the absorbance values of MCF-7 cells in BC-MSC group and BC-MSC+IL-6 neutralizing antibody group were 0.56±0.05 and 0.42±0.04, respectively, and the difference was statistically significant ( t = -3.11, P < 0.05); the results of Transwell assay showed that the numbers of membrane-penetrating cells in the two groups were 30.3±1.5 and 17.3±2.1, respectively, and the difference was statistically significant ( t = -7.12, P < 0.05); qRT-PCR assay showed that the relative expressions of E-cadherin mRNA were 0.44±0.05 and 0.76±0.05 ( t = 6.40, P < 0.01), the relative expressions of vimentin mRNA were 2.90±0.21 and 1.79±0.21 ( t = 5.29, P < 0.01), and the relative expressions of snail mRNA were 3.20±0.20 and 1.91±0.30 ( t = 2.16, P < 0.01); Western blotting assay showed that the degrees to down-regulate the expression of E-cadherin protein and up-regulate the expressions of vimentin and snail proteins in the BC-MSC+IL-6 neutralizing antibody group were weakened compared with the BC-MSC group. Conclusions:BC-MSC can promote the proliferation and migration of breast cancer MCF-7 cells probably through activating IL-6-STAT3 signaling pathway-induced EMT by its secretion of IL-6.
