LncRNA WT1-AS Inhibits Triple-Negative Breast Cancer Cell Migration and Invasion by Downregulating Transforming Growth Factor β1

TNBC patients

This study passed the review board of Shanxi Provincial People's Hospital. Research subjects of this study were 62 TNBC patients (33–67 years; mean = 49.7 ± 4.9 years), who were selected from the 114 TNBC cases diagnosed and treated in the aforementioned hospital. Inclusion criteria were as follows: (1) no therapies were initiated within 3 months; and (2) newly diagnosed TNBC cases. Exclusion criteria were as follows: (1) patients complicated with other clinical disorders; (2) recurrent TNBC; (3) therapies were performed; and (4) history of malignancies. AJCC staging system was used to stage the 62 patients. Based on clinical findings, the 63 patients included 12, 20, 18, and 12 cases at stages I–IV, respectively. The patients were informed of the details of experimental design. They provided informed consent for the potential publication of the experimental data derived from this study.

TNBC tissue samples and cells

Breast biopsy under the guidance of magnetic resonance imaging was performed on all the 62 TNBC patients before the initiation of therapies. During biopsy, TNBC and nontumor tissue samples were collected (0.018–0.026 g). Histopathological examinations were performed to test all tissue samples. All nontumor tissue samples contained <1% cancer cells, and all TNBC tissue samples contained >95% cancer cells.

BT-549 human TNBC cell line (ATCC) was used. To prepare cell culture medium, RPMI-1640 medium was mixed with fetal bovine serum (FBS) in a ratio of 9:1. Cell culture conditions were 37°C, 5% CO₂, and 95% humidity.

Transient cell transfection

BT-549 cells were harvested at 70%–80% confluence to perform transfections. pcDNA3.1 vector was used to construct WT1-AS and TGF-β1 expression vectors, and the vector construction service was provided by Sangon (Shanghai, China). Negative control (NC) siRNA and WT1-AS siRNA were from Sangon. All transient transfections were mediated by Lipofectamine 2000 (Invitrogen) to transfect 10 nM vectors (empty vector as NC group) or 40 nM siRNAs (NC siRNA as NC group) into 3 × 10⁶ cells. Control (C) group for all transfections was untransfected cells. Transfections were performed in three biological replicates. At 24 h post-transfection, cells were harvested and used for subsequent experiments (significant silencing started from 12 h post-transfection and lasted until 100 h post-transfection).

RNA extractions and qPCR

Total RNAs in 3 × 10⁵ BT-549 cells (collected at 24 h post-transfection) or 0.015 g tissue samples (ground in liquid nitrogen) were extracted using RNAzol reagent (Sigma-Aldrich). Tetro Reverse Transcriptase (Bioline) was used to perform all reverse transcriptions. To measure the expression levels of WT1-AS and TGF-β1 mRNA, KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) was used to prepare all qPCR mixtures with GAPDH as endogenous control. PCRs were performed in three replicates and 2^−ΔΔCT method was used to normalize Ct values.

Western blot

Total proteins in 3 × 10⁵ BT-549 cells (collected at 24 h post-transfection) were extracted by RIPA solution (Thermo Fisher Scientific) and quantified by bicinchoninic acid assay (Thermo Fisher Scientific). Before electrophoresis (10% sodium dodecyl sulfate – polyacrylamide gel electrophoresis), all protein samples were denatured by incubating with boiling water for 5 min. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, followed by blocking with 5% nonfat milk at 24°C for 90 min. After that, anti-TGF-β1 (1:1500, ab50716; Abcam) and anti-GAPDH (1:1500, ab37168; Abcam) rabbit primary antibodies were used to incubate with membranes for 18 h at 4°C. Then, immunoglobulin G–horseradish peroxidase (1:1000, MBS435036; MyBioSource) goat secondary antibody was used to incubate with the membranes for another 2 h at 24°C. Finally, ECL Western Blotting Substrate (Thermo Fisher Scientific) was used to incubate with membranes for 10 min at 24°C to develop signals. ImageJ v1.48 software was used to normalize data.

Transwell assays

Single-cell suspensions were prepared by mixing 1 mL mixture of 90% RPMI-1640 medium and 10% FBS with 3 × 10⁴ cells. Effects of transfections on the invasion and migration of BT-549 cells were analyzed using Transwell assay. To mimic in vitro cell invasion, Matrigel (200 mg/mL, 356234; Millipore) was used to coat up chamber membranes. The upper chamber was filed with cell suspension, and the lower chamber was filled with a mixture of 80% RPMI-1640 medium and 20% FBS. Cells were cultivated under aforementioned conditions for 12 h. After staining with 0.5% crystal violet (Sigma-Aldrich) for 2 h at 24°C, cells were counted under a light microscope. The numbers of invading and migrating cells were normalized to cell proliferation rates (OD values measured at 450 nM after the addition of CCK-8 solution) at 12 h after the initiation of experiments.

Statistical analysis

All experiments were performed in three biological replicates. Mean values were calculated and were used for all cell analyses. Correlations were analyzed by linear regression. Differences were explored between two types of tissues or among multiple patient and cell groups by performing paired t test and analysis of variance (ANOVA) (one-way) in combination with Tukey test, respectively. p < 0.05 was statistically significant.

WT1-AS was downregulated in TNBC and was affected by clinical stages

qPCR and unpaired t test were performed to measure and compare the expression levels of WT1-AS in nontumor and TNBC tissues. Compared with nontumor tissues, significantly lower expression levels of WT1-AS were observed in TNBC tissues (Fig. 1A, p < 0.05). ANOVA (one-way) in combination with Tukey test was used to compare expression levels of WT1-AS in TNBC tissues among different clinical stages. It can be observed that expression levels of WT1-AS decreased significantly with the increase in clinical stages (p < 0.05; Fig. 1B).

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FIG. 1.

WT1-AS was downregulated in TNBC and was affected by clinical stages. qPCR and unpaired t test were performed to measure and compare the expression levels of WT1-AS in nontumor and TNBC tissues (A). ANOVA (one-way) in combination with Tukey test was used to compare expression levels of WT1-AS in TNBC tissues among different clinical stages (B). Experiments were performed in three replicates, *p < 0.05. ANOVA, analysis of variance; TNBC, triple-negative breast cancer.

TGF-β1 mRNA and WT1-AS were inversely correlated in TNBC tissues

qPCR and unpaired t test were performed to measure and compare the expression levels of TGF-β1 mRNA in nontumor and TNBC tissues. Compared with nontumor tissues, significantly higher expression levels of TGF-β1 mRNA were observed in TNBC tissues (p < 0.05; Fig. 2A). The correlation between TGF-β1 mRNA and WT1-AS was analyzed by linear regression. It can be observed that expression levels of TGF-β1 mRNA were significantly and inversely correlated with the expression levels of WT1-AS in TNBC tissues (Fig. 2B). However, the correlation between them was not significant in nontumor tissues (Fig. 2B).

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FIG. 2.

TGF-β1 mRNA and WT1-AS were inversely correlated in TNBC tissues. qPCR and unpaired t test were performed to measure and compare the expression levels of TGF-β1 mRNA in nontumor and TNBC tissues (A). The correlation between TGF-β1 mRNA and WT1-AS in TNBC (B) and nontumor (C) tissues was analyzed by linear regression. Experiments were performed in three replicates, *p < 0.05. TGF-β, transforming growth factor β.

WT1-AS negatively regulated the expression of TGF-β1 in BT-549 cells

WT1-AS and TGF-β1 vectors and WT1-AS siRNA were transfected into BT-549 cells. Expression levels of WT1-AS and TGF-β1 were measured at 24 h post-transfection by qPCR. Compared with two controls C and NC, expression levels of WT1-AS and TGF-β1 mRNA were significantly altered (Fig. 3A, p < 0.05). Moreover, compared with two controls, WT1-AS expression was not significantly altered after TGF-β1 overexpression (Fig. 3B). However, WT1-AS overexpression led to downregulated expression, whereas WT1-AS siRNA silencing led to upregulated expression of TGF-β1 at both mRNA and protein levels (p < 0.05; Fig. 3C).

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FIG. 3.

WT1-AS negatively regulated the expression of TGF-β1 in BT-549 cells. WT1-AS and TGF-β1 vectors and WT1-AS siRNA were transfected into BT-549 cells. Transfections were confirmed by qPCR at 24 h post-transfection (A). The effects of TGF-β1 overexpression on WT1-AS expression were also analyzed by qPCR (B). The effects of WT1-AS overexpression on TGF-β1 expression at both mRNA and protein levels were analyzed by qPCR and Western blot, respectively (C). Experiments were performed in three replicates, p < 0.05. TGF-β, transforming growth factor β. *p < 0.05.

WT1-AS suppressed BT-549 cell invasion and migration through TGF-β1

Transwell assays were performed to analyze the effects of transfections on the invasion (Fig. 4A) and migration (Fig. 4B) of BT-549 cells. Compared with two controls C and NC, WT1-AS overexpression caused inhibited invasion and migration of TNBC cells. TGF-β1 overexpression and WT1-AS siRNA silencing showed opposite functions and TGF-β1 overexpression reduced the effects of WT1-AS overexpression on cell behaviors (p < 0.05).

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FIG. 4.

WT1-AS suppressed BT-549 cell invasion and migration through TGF-β1. Transwell assays were performed to analyze the effects of transfection of WT1-AS and TGF-β1 expression vectors and WT1-AS siRNA on the invasion (A) and migration (B) of BT-549 cells. Experiments were performed in three replicates, p < 0.05. TGF-β, transforming growth factor β. *p < 0.05.