Figures & data
Figure 1 Zinc finger protein 259 (ZNF259) was upregulated in breast cancer tissues and related to poor prognosis.
Notes: (A) Data from KM Plotter Online Tool showed that ZNF259 expression significantly correlated with breast cancer patients’ poor survival (RFS, n=3,951, P=7.1×10−8 OS, n=1,402, P=1.5×10−5; DMFS, n=1,746, P=0.00028). (B) ZNF259 was very weakly stained in normal breast duct glandular epithelium cells and myoepithelial cells. (C) ZNF259 was moderately positive in the cytoplasm of breast cancer cells. (D) ZNF259 showed strong positive staining in the cytoplasm of cancer cells, and scattered nucleus-stained cells could also be observed although not very obviously. (E) ZNF259 expression in breast cancer cells was much higher than that in adjacent normal breast duct glandular epithelium cells in the same field of view (400× and partial enlargement).
Abbreviations: DMFS, distant metastasis-free survival; OS, overall survival; RFS, relapse-free survival; ZNF259, zinc finger protein 259.
Table 1 ZNF259 expression in adjacent normal breast tissue and breast cancer tissue
Table 2 The relevance of ZNF259 expression to clinicopathological characteristics of breast cancer patients
Figure 2 ZNF259 expression increased in breast cancer tissues and cell lines.
Notes: (A) Western blotting was performed on 12 pairs of fresh breast cancer tissues and adjacent nontumor tissues. ZNF259 expression in most breast cancer tissues was higher than that in the corresponding adjacent nontumor tissues (11/12, 91.67%). (B) The relative expression ratio of ZNF259 in breast cancer tissues was significantly higher than that in the adjacent nontumor tissues (bars indicate the mean ± standard error of the mean, ***P<0.001). (C, D) ZNF259 expression in the breast cancer cell lines T47D, MCF-7, BT-549, MDA-MB-231, and MDA-MB-468 was significantly higher than that in the normal human mammary epithelial cell MCF-10A (bars represent the mean ± standard deviation, ***P<0.001).
Abbreviation: ZNF259, zinc finger protein 259.
Figure 3 ZNF259 knockdown inhibited the invasion and migration abilities of breast cancer cells.
Notes: (A, B) ZNF259 was knocked down by siRNAs in MCF-7 and MDA-MB-231 cell lines (mean ± standard deviation, **P<0.01). (C, D) Transwell analysis showed that ZNF259 knockdown inhibited invasion by MCF-7 and MDA-MB-231 cells (mean ± standard error of the mean, *P<0.05). (E, F) Wound healing assay revealed that silencing ZNF259 expression also inhibited migration by MCF-7 and MDA-MB-231 cells (mean ± standard error of the mean, *P<0.05; **P<0.01).
Abbreviation: ZNF259, zinc finger protein 259.
Figure 4 ZNF259 knockdown could downregulate phosphorylated-extracellular signal-regulated kinase (p-ERK), phosphorylated-glycogen synthase kinase 3β (p-GSK3β), and Snail expression, and upregulate E-cadherin and zonula occludens protein 1 (ZO-1) expression.
Notes: Western blotting was performed after ZNF259 knockdown in both MCF-7 and MDA-MB-231 cell lines, and phosphorylated mitogen-activated protein kinase (p-MEK), phosphorylated-extracellular signal-regulated kinase (p-ERK), phosphorylated-glycogen synthase kinase 3β (p-GSK3β) (Ser 9), and Snail expressions were downregulated, while E-cadherin and ZO-1 expression was upregulated. The total MEK, ERK, and GSK3β levels did not show obvious changes. The numbers indicated the relative expression ratios of the protein. At least 3 separate experiments were performed. The statistical analysis histogram is shown in the .
Abbreviation: ZNF259, zinc finger protein 259.
Figure 5 Invasion and migration promoted by ZNF259 were inhibited by the extracellular signal-regulated kinase (ERK) inhibitor U0126.
Notes: (A) ZNF259 transfection could upregulate p-ERK, phosphorylated-glycogen synthase kinase 3β (p-GSK3β), and Snail expression, and downregulate E-cadherin and zonula occludens protein 1 (ZO-1) expression. All these effects could be restored by the incorporation of the ERK inhibitor U0126 (5 µM). The numbers indicated the relative expression ratios of protein. At least 3 separate experiments were performed. (B, C) Transwell assay showed that U0126 could reverse the ZNF259 transfection-caused increase in cell invasion ability (mean ± standard error of the mean, **P<0.01). (D, E) Wound healing assay also showed that U0126 could reverse the ZNF259 transfection-caused increase in cell migration ability (mean ± standard error of the mean, *P<0.05).
Abbreviation: ZNF259, zinc finger protein 259.
Figure S1 Statistical analysis histogram of the Western blotting results for zinc finger protein 259 (ZNF259) knockdown in both MCF-7 and MDA-MB-231 cell lines.
Notes: At least 3 separate experiments were performed. In both the MCF-7 and MDA-MB-231 cell line, ZNF259 knockdown could significantly downregulate phosphorylated mitogen-activated protein kinase (p-MEK), phosphorylated-extracellular signal-regulated kinase (p-ERK), phosphorylated-glycogen synthase kinase 3β (p-GSK3β), and Snail expression, and upregulate E-cadherin and zonula occludens protein 1 (ZO-1) expression. The data are shown as mean ± standard deviation (SD), *P<0.05, **P<0.01, ***P<0.001.
Abbreviation: ZNF259, zinc finger protein 259.
