NEAT1 negatively regulates miR-218 expression and promotes breast cancer progression

2.1 Patients and tissue samples

Seventy paired breast cancer and corresponding adjacent normal tissue specimens were collected from surgical resections performed at Binzhou Medical University Hospital, snap-frozen at the time of surgery, and stored at - 80 ∘ C until further analysis. No patients received chemotherapy or radiotherapy prior to surgery. Written informed consent was obtained from all patients. This study was approved by the Ethics Committee of Binzhou Medical University Hospital.

2.2 Cell culture

Two human breast cancer cell lines (MCF-7 and SK-BR-3) and an immortalized breast epithelial cell line (MCF-10A) were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) and maintained at 37 ∘ C in a 5% CO 2 atmosphere.

2.3 Cell transfection

Si-NEAT1-1, si-NEAT1-2, si-negative control (NC), and miR-218 inhibitor were purchased from GenePharma Co., Ltd. (Shanghai, China). The sequences were as follows: siRNA NEAT1-1, 5’-GCCA UCAGCUUUGAAUAAAUU-3’; and siRNA NEAT1-2, 5’-GGUGUUAUCAAGUGAAUUAUU-3’. Cells were transfected using Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

2.4 Cell proliferation and cell colony formation assays

Cell proliferation was detected using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) following the manufacturer’s instructions. MCF-7 and SK-BR-3 cells transfected with si-NC, si-NEAT1-1, or si-NEAT1-1 + miR-218 inhibitor were plated in 6-well plates 1, 2, 3 and 4 days after transfection. Briefly, the CCK-8 solution (10% of the medium [10 μ l]) was added to each well and incubated for 4 h prior to analysis. Then, the absorbance at 450 nm was measured. A cell colony formation assay was performed in 6-well plates. Two hundred transfected cells/well were seeded and cultured for 2 weeks, then the cells were fixed in methanol and stained with 0.1% crystal violet and the number of cell colonies were calculated.

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For cell invasion assays, 1.0 × 10 5 MCF-7 and SK-BR-3 cells were plated into the upper Transwell chamber (8.0 μ m; BD Biosciences, San Jose, CA, USA) for cell invasion assays. Forty-eight hours after transfection, the upper Transwell chamber cells were removed, and the cells on the underside of the membrane were fixed in 100% methanol, then stained with 0.1% crystal violet. The number of stained cells in the chambers was determined.

2.6 Quantitative real time-PCR (qRT-PCR)

Total RNA was collected using TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, USA) and reverse-transcribed to cDNA using a Reverse Transcription Kit (TAKAKA, Japan). qPCR was carried out using SYBR Green PCR master mix (Bio-Rad, Hercules, CA, USA) GAPDH and U6 were used as internal controls. Quantitative RT-PCR was performed on the Roche LightCycler ® 96 (LC96) real-time PCR platform using the 2 - Δ ⁢ Δ ⁢ CT method. The primer sequences were as follows: NEAT1 forward, 5’-CTTCCTCCCTTTAACTTATCCATTCAC-3’ and reverse, 5’-CTCTTCCTCCACCATTACCAACAATAC-3’; and miR-218 forward, 5’-AAGACACCCTGGACG AAGCC-3’ and reverse, 5’-ACAACCAGAGTCCACC GGCG-3. Each experiment was performed in triplicate.

2.7 Dual-luciferase reporter assay

MCF-7 cells were co-transfected with 150 ng of pmirGLO-NEAT1-WT or pmirGLO-NEAT1-MUT and miR-218 mimic (Ribobio, Guangzhou, China). Two nanograms of pRL-TK (Promega, Madison, WI, USA) were also co-transfected with miR-218 mimic or miR-NC into MCF-7 cells using Lipofectamine 3000 (Invitrogen). The relative luciferase activity was normalized to Renilla luciferase activity 48 h after transfection. Each experiment was repeated at least three times.

2.8 Statistical analyses

Statistical analyses were carried out by using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). Differences among variables were analyzed using a two-tailed Student’s t-test. Data are presented as the mean ± SD. 0.05 was defined as statistical significance.

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3.1 Expression of NEAT1 is increased in breast cancer tissues and is associated with poor prognosis

To identify the level of NEAT1 expression in breast cancer, we compared the expression of NEAT1 mRNA in tumor specimens and adjacent normal tissues. NEAT1 was significantly increased in breast cancer tissues compared with adjacent normal tissues (Fig. 1A). Furthermore, we analyzed the association between NEAT1 expression and clinicopathologic data. As shown in Table 1, the expression of NEAT1 was associated with TNM stage ( P = 0.014) and lymph node metastasis ( P = 0.03). The expression of NEAT1, however, was not associated with age ( P = 0.816), tumor size ( P = 0.871), and HER-2 status ( P = 0.632; Table 1). Kaplan-Meier analysis showed that higher NEAT1 in breast patients predicted a poor overall survival compared with lower NEAT1 expression (Fig. 1B [log-rank = 11.019]; 0.05). Thus, increased expression of NEAT1 was a predictor of prognosis for breast cancer patients.

3.2 MiR-218 is a direct target of NEAT1 in breast cancer cells

Next, we determined the expression of NEAT1 in two breast cancer cell lines (MCF-7 and SK-BR-3), and an immortalized breast epithelial cell line (MCF-10A). The expression of NEAT1 was also increased in breast cancer cells compared with MCF-10A (Fig. 2A). When NEAT1 was knocked down by two siRNA oligos in MCF-7 and SK-BR-3 cells, we showed that si-NEAT1-2 had higher knockdown efficiency. Thus, we selected si-NEAT1-2 for the following experiments (Fig. 2B and C).

According to bioinformatics analysis using miRanda (www.microrna.org), a novel miR-218 binding site existed with NEAT1 (Fig. 3A). Furthermore, we analyzed the level of miR-218 expression in breast cancer tissues, which was significantly down-regulated compared with the adjacent normal tissues (Fig. 3B). The Pearson correlation coefficient indicated that there was a negative association between NEAT1 and miR-218 expression (Fig. 3C). The dual-luciferase reporter assay was performed to verify that miR-218 is a functional target of NEAT1 in breast cells. MiR-218 mimic reduced the luciferase activity of pmirGLO-NEAT1-WT, whereas miR-218 mimic did not affect the luciferase activity of pmirGLO-NEAT1-MUT in MCF-7 cells (Fig. 3D). Thus, miR-218 was a direct target of NEAT1. In addition, we also demonstrated that miR-218 expression was up-regulated after NEAT1 silencing in MCF-7 cells (Fig. 3E). NEAT1 expression was up-regulated after miR-218 inhibitor was transfected into MCF-7 cells, but NEAT1 expression was down-regulated after miR-218 mimic was transfected into MCF-7 cells (Fig. 3F). Thus, NEAT1 mediated regulation of mir-218 in breast cancer cells.

3.3 NEAT1 mediates regulation of miR-218 expression to promote cell proliferation and invasion in breast cancer cells

Having verified that miR-218 is a direct target of NEAT1, we further investigated the effects of NEAT1 expression on cell proliferation and invasion. Cell proliferation was dramatically inhibited after transfecting si-NEAT1 into MCF-7 or SK-BR-3 cells, but co-transfection of si-NEAT1 and miR-218 inhibitor counteracted the effects compared with si-NC based on the CCK8 assay (Fig. 4A and B). The cell colony formation assay showed that the number of cell colonies was dramatically reduced after transfecting si-NEAT1 into MCF-7 or SK-BR-3 cells, but co-transfection of si-NEAT1 and miR-218 inhibitor counteracted the effects (Fig. 4C and F). Cell invasion was assessed using the Transwell cell invasion assay, which showed that cell invasion was inhibited after transfecting si-NEAT1 into MCF-7 or SK-BR-3 cells, but co-transfection of siNEAT1 and miR-218 inhibitor counteracted the effects compared with si-NC (Fig. 5A–D). Thus, NEAT1 negatively regulates miR-218 promotion of cell proliferation and invasion.