MicroRNAs (miRNAs) are small non-coding RNAs that are key post-transcriptional regulators of gene expression. MicroRNA-214 (miR-214) and microRNA-218 (miR-218) have shown the function of tumor suppressors in various types of human cancers. However, the biological functions of miR-214 and miR-218 in breast cancer have not been elucidated completely. The present study evaluated the expression and biological function of miR-214 and miR-218 in human breast cancer. Our results revealed that the expression of miR-214 and miR-218 were significantly decreased in breast cancer tissues compared with adjacent tissues. The aberrant expression of miR-214 and miR-218 were negatively associated with Ki-67, and the miR-218 expression was positively associated with progesterone receptor (PR) in breast cancer tissues.
Breast cancer is one of the most common malignancies among females (
MicroRNA (miRNA) is a class endogenous non-coding single-stranded RNA. As circulating marker, miRNA is being studied extensively (
In the present study, we investigate the expression of miR-214 and miR-218 in breast cancer and adjacent tissues, and analyzed the correlations in miR-214 and miR-218 expression and the clinicopathological characteristics. The effects of miR-214 or miR-218 on cell proliferation, apoptosis and cell cycle were also determined
Forty-nine breast cancer tissues and their paired adjacent tissue samples, which were diagnosed by pathological surgical resection, were collected between 2013 and 2015 at The First Hospital of Hebei Medical University (Shijiazhuang, China). The tissues were frozen in liquid nitrogen at −80°C immediately until use. Breast cancer patients who had undergone chemotherapy or radiation therapy before surgery were excluded.
Permission to use human tissue samples for research purposes was approved by the Biomedical Ethics Committee of Hebei Medical University, Shijiazhuang, Hebei, China. All patients were female and consented to participate in the present study.
Breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), cultured in RPMI-1640 containing 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 mg/ml of streptomycin (Gibco, Grand Island, NY, USA) in a humidified atmosphere containing 5% CO2 at 37°C. For transfection, cells were seeded and cultured for 24 h in 12-well plates. According to the manufacturer's instructions, cells were transfected with miR-214 mimic, miR-218 mimic or negative control, respectively, by Lipofectamine 2000 (Invitrogen Life Technologies, Grand Island, NY, USA) in serum-free medium. Six hours after the transfection, the complete medium was changed and maintained for 48 h at 37°C in 5% CO2. The mimics of miR-214, miR-218 and negative control were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China).
Total RNA was extracted from breast cancer tissues, adjacent tissues and MCF-7 cells by TRIzol reagent (Invitrogen), according to the manufacturer's instructions. Reverse transcription PCR and real-time PCR were performed with TaqMan microRNA Reverse Transcription kit and TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA, USA, respectively) following standard protocol. U6 was used as an endogenous control to normalize variance. The primers of miR-214, miR-218 and U6 were purchased from Applied Biosystems. The fold changes were calculated via relative quantification (2−ΔCT).
Cell proliferation was determined by the Cell Counting kit-8 assay (CCK-8; Dojindo Molecular Technologies, Inc., Beijing, China). MCF-7 cells (1,000 cells/well) were cultured in 96-well plates. After incubation, 10
The effects of miR-214 and miR-218 on breast cancer cell apoptosis and cell cycle were examined by flow cytometry (BD Biosciences, Mansfield, MA, USA). In brief, MCF-7 cells were transfected with miR-214 mimic, miR-218 mimic or negative control for 48 h, the cells were completely collected including apoptotic cells in culture medium, and washed twice with cold PBS. The cell apoptosis were analyzed by Annexin V-FITC detection kit (Neobioscience Technology, Shenzhen, China) according to the manufacturer's protocol. For determining the cell cycle, the cells were transfected for 48 h, washed twice with PBS, treated with trypsin, and fixed with 75% ethanol overnight at −20°C, and incubated with 100 mg/ml RNase A and 50 mg/ml propidium iodide (PI) at room temperature for 30 min. The percentage of the G0/G1, S and G2/M populations were evaluated in each group. The data were analyzed by CellQuest Pro software. Each experiment was performed in triplicate.
MCF-7 cells were grown to confluence. A wound was made by scraping with a conventional pipette tip across the confluence cell layer, and washed with PBS twice. Forty-eight hours after scraping, migration was determined, using the ImageJ, as a percentage of healing area relative to the initial wound area.
Data were processed by the SPSS Graduate Pack 13.0, GraphPad 5 software or the Student's t-test. P<0.05 was considered to be statistically significant.
To assess the effects of miR-214 and miR-218 on breast cancer development, we first evaluated miR-214 and miR-218 expression levels in breast cancer and adjacent cancer tissues by real-time RT-PCR. The expression of miR-214 and miR-218 was significantly downregulated in breast cancer tissues compared with matching adjacent tissues (P<0.01, respectively;
We further evaluated the correlation of miR-214 or miR-218 expression and the clinicopathological factors, including age, tumor size, lymph node metastasis, clinical stage, estrogen receptor (ER), progesterone receptor (PR), epidermal factor receptor-2 (HER-2), p53 and Ki-67. ER, PR, HER-2, p53 and Ki-67 were detected by immunohistochemistry, respectively. When the number of stained cells was >10% in one field, it was defined as positive. We found that miR-214 and miR-218 expression was negatively associated with Ki-67 expression (
To test the biological function of miR-214 and miR-218, the miR-214 mimic, miR-218 mimic and negative controls were purchase from Applied Biosystems. The overexpression of miR-214 or miR-218 was carried out in MCF-7 cells by transfection with miR-214 mimic or miR-218 mimic, respectively. The expression of miR-214 (
The cell cycles were analyzed by flow cytometry (BD Biosciences) after staining with propidium iodide (PI). The percentage of cells in the G0/G1, S and G2/M phases was, respectively, evaluated under the negative control (
The cells were seeded into 6-well plates and transfected with negative control, miR-214 or miR-218 mimic, and cell apoptosis were analyzed by Annexin V-FITC detection kit according to the manufacturer's protocol. Representative results of negative control (
Cell migration was tested by wound-healing assay. Cell migration was decreased in miR-214 mimic (28.02±0.89 vs. 44.38±3.71%, P=0.013) and miR-218 (27.07±5.75 vs. 44.38±3.71%, P=0.017) mimic treated cells compared with negative control transfected cells (
The expression of miR-214 has been verified downregulated in human cervical cancer (
In particular, expression of miR-214 and miR-218 was reduced in human breast cancer tissues, and miR-214 or miR-218 expression is negatively associated with Ki-67, a cellular marker of tumor cells proliferation (
Our results also prove that the miR-214 and miR-218 promote early apoptosis of breast cancer cells (
The effects of miR-214 and miR-218 on breast cancer cell migration are shown in
In conclusion, our results show that miR-214 and miR-218 expression are decreased in breast cancer tissues. Overexpression of miR-214 or miR-218 could suppress cell proliferation and migration, disturb the cell cycle and induce cell apoptosis
The present study was supported by the Science and Technology Support Project of Hebei, China (no. 14277755D).
The expression of miR-214 and miR-218 were decreased in breast cancer tissues. The (A) expression of miR-214 and (B) miR-218 was detected in breast cancer tissues and adjacent tissues of 49 patients by real-time RT PCR. The P-values were assessed by non-parametric test.
Correlation analysis of miR-214 and miR-218, respectively, and clinicopathological data. The correlation between (A and B) miR-214 or (C and D) miR-218 expression and Ki-67 or progesterone receptor (PR) were evaluated, respectively. Ki-67 and PR were detected by immunohistochemistry, when the number of stained cells was >10% in one field, it was defined as positive. The P-values were assessed by non-parametric test. NS, indicates no statistical difference.
Overexpression of miR-214 and miR-218 inhibits breast cancer cell proliferation. (A) expression of miR-214 and (B) miR-218 was upregulated with the transfection of miR-214 mimic or miR-218 mimic in MCF-7 cells, respectively. (C) Forty-eight hours after transfection, the cell proliferation was determined by CCK-8 assay. The experiments were performed in triplicate. #P=0.0253 (miR-214 mimic transfected cells vs. negative control cells). *P=0.0282 (miR-218 mimic transfected cells vs. negative control cells).
Effects of miR-214 and miR-218 on the cell cycle of breast cancer. After transfection, the cells were washed with PBS twice, and treated with trypsin. The surviving cells were collected, excepting apoptotic cells, in culture medium. The cell cycles were analyzed by flow cytometry after staining with PI. The representative results of cell cycles are shown in (A) negative control, (B) miR-214 mimic and (C) miR-218 mimic transfected cells. (D) In addition, the percentage of cells in the G0/G1, S and G2/M phases was, respectively, evaluated. The experiments were performed in triplicate. *P=0.0298 (the percentage of S phase cells, miR-214 mimic transfected cells vs. negative control cells). &P=0.0009 (the percentage of S phase cells, miR-218 mimic transfected cells vs. negative control cells). #P=0.006 (the percentage of G0/G1 phase cells, miR-218 mimic transfected cells vs. negative control cells).
Effects of miR-214 and miR-218 on apoptosis of breast cancer cells. After (A) negative control, (B) miR-214 mimic and (C) miR-218 mimic transfection, the cells were collected including apoptotic cells in culture medium. In addition, cell apoptosis was analyzed by Annexin V-FITC detection kit. (A–C) Representative results are shown. (D) The percentage of early apoptotic cells was calculated, respectively. The experiments were performed in triplicate. The P-values were assessed by the Student's t-test.
Effects of miR-214 and miR-218 on migration capability of breast cancer cells. After negative control, miR-214 mimic and miR-218 mimic transfection, the cell migration was measured by wound-healing assay. (A) Representative results are shown. (B) The percentage of healing area was determined using the ImageJ. The experiments were performed in triplicate. The P-values were assessed by the Student's t-test.
Correlations of miR-214 and miR-218 expression and the clinicopathological characteristics of breast cancer patients.
Parameters | No. of patients | Relative miR-214 expression Median (interquartile range) | P-value | Relative miR-218 expression Median (interquartile range) | P-value |
---|---|---|---|---|---|
Age (years) | 0.27 | 0.81 | |||
≤52 | 25 | 104.02 (26.96–689.51) | 43.73 (10.42–179.10) | ||
>52 | 24 | 116.64 (26.30–221.89) | 47.39 (13.84–144.50) | ||
Tumor size (cm) | 0.82 | 0.63 | |||
≤2 | 17 | 122.86 (25.15–427.12) | 30.29 (5.10–248.76) | ||
>2 | 32 | 107.23 (29.92–246.91) | 47.38 (18.90–100.79) | ||
Lymph node metastasis | 0.90 | 0.94 | |||
Negative | 23 | 110.43 (26.69–406.72) | 50.74 (15.88–94.53) | ||
Positive | 26 | 94.33 (26.87–294.63) | 43.88 (12.79–170.83) | ||
Clinical stage |
0.77 | 0.72 | |||
I | 11 | 124.65 (23.62–447.52) | 81.16 (7.47–303.16) | ||
II, III | 38 | 107.23 (27.63–253.54) | 43.88 (15.25–119.50) | ||
ER | 0.77 | 0.32 | |||
Negative | 13 | 122.86 (37.19–160.50) | 26.69 (10.39–160.97) | ||
Positive | 36 | 90.27 (26.04–437.32) | 57.13 (18.77–161.17) | ||
Her-2 | 0.82 | 0.68 | |||
Negative | 20 | 65.41 (17.21–698.63) | 35.18 (9.21–161.17) | ||
Positive | 29 | 123.76 (38.05–255.90) | 50.74 (16.05–163.83) | ||
p53 | 0.29 | 0.16 | |||
Negative | 33 | 65.81 (22.50–333.40) | 40.06 (9.82–132.00) | ||
Positive | 16 | 124.21 (48.80–398.44) | 58.11 (24.42–329.89) |
No stage IV patients. ER, estrogen receptor; PR, progesterone receptor; HER-2, epidermal growth factor receptor-2.