lncRNA ACTA2-AS1 predicts malignancy and poor prognosis of triple-negative breast cancer and regulates tumor progression via modulating miR-532-5p

Study subjects

There were 119 patients with TNBC diagnosed at Fujian Provincial Maternity and Children’s Hospital enrolled in this study from January 2013 to December 2015. The inclusion and exclusion criteria were: 1) patients with negative expression of ER, PR, and HER2; 2) patients were primarily diagnosed with TNBC; 3) patients received surgery for the first time; 4) the clinical data of enrolled patients are completed; 5) patients had received adjuvant therapy before surgery were excluded. The study had been approved by the Ethics Committee of Fujian Provincial Maternity and Children’s Hospital (approval no. 2013188) and had obtained signed informed consent from patients.

The included patients received surgical treatment and the tissue samples (tumor and normal paracancerous tissues) were collected during the surgery. The tissues were confirmed by at least two pathologists (ER, PR < 1% and non-amplified HER-2 on FISH) and stored at − 80 °C. The tumor nuclei and necrosis were assessed with tumor sections.

Follow-up survey

All patients were followed-up for 5 years after their surgical treatments through outpatient clinical review or telephone. The recurrence, metastasis, and death events were recorded as the end events, and unrelated death was excluded.

Cell culture

TNBC cell lines (MDA-MB-231, HCC-1937, Hs578t, and BT-20) and a normal cell line MCF-10A were purchased from America Typical culture collection (ATCC, USA) and cultured according to standard protocols. Briefly, the cell culture was carried out in the DMEM medium (catalog# 11995065, Gibco, USA) with 10% FBS (catalog# 10438034, Thermo Fisher Scientific, USA) and 1 × 105 U/L penicillin and streptomycin (catalog# 15070063, Gibco, USA) under sterile conditions at 37 °C with 5% CO2.

Cell transfection

The cultured cells at the logarithmic growth stage with a density of 70–80% confluence were used in the cell transfection. To regulate ACTA2-AS1, cells were transfected with pcDNA 3.1-ACTA2-AS1 (oe-ACTA2-AS1) or corresponding negative controls (oe-NC). pcDNA3.1 and negative controls were obtained from Life Technologies (USA) and the transfected concentration was 50 nM. While miR-532-5p mimic, inhibitor, or negative controls (50 nM) was transfected into cells to regulate miR-532-5p. Cell transfection was conducted with the help of Lipofectamine 2000 (catalog# 11668019, Invitrogen, USA) for 48 h, and then the cells were washed and collected for the following analyses.

Real-time qPCR

Total RNA was first isolated from cell and tissue samples using TRIzol (catalog#15596018, Invitrogen, USA) according to the manufacturer’s protocols. The purity and concentration of extracted RNA were evaluated by the absorbance at 260 and 280 nm. The reverse transcription into cDNA was performed with 2 μg RNA and the lnRcute lncRNA First-standard cDNA kit (for ACTA2-AS1, catalog# KR202, TIANGEN, China) or the miRcute miRNA First-standard cDNA Synthesis kit (for miR-532-5p, catalog# KR201, TIANGEN, China) according to the manufacturer’s instruments. Then, the PCR amplification was performed with the SYBR Green kit on the Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, Germany) according to the following reaction conditions: 95 °C × 10 min; (95 °C × 30 s, 50 °C × 1 min, 72 °C × 1 min) for 20 cycles; 72 °C × 3 min. The relative expression levels of ACTA2-AS1 and miR-532-3p were calculated with the 2−ΔΔCt method normalized to GAPDH and cel-miR-39, respectively. The sequences of used primers are summarized in Table S1.

Cell proliferation assessment

Cells (5 × 105 cells/well) were planted in the 96-well plates and maintained in the DMEM culture medium (containing 10% FBS) for a certain period. Then, the cells were washed with PBS and resuspended in the culture medium. CCK8 (catalog# CK04, Dojindo, Japan) was added to each well with a ratio of 1: 10 to the culture medium and incubated for another 2 h. The absorbance at 450 nm was detected with a microplate reader (Molecular Devices, USA).

Cell metastasis assessment

Cells (5 × 105 cells/well) were seeded into the upper chamber of the Transwell plates (24-well with a pore size of 8 μm) maintained with an FBS-free culture medium at 37 °C with 5% CO2 for 48 h and allowed across the chamber plates. The Matrigel (catalog# 356234, corning, USA) was used to coat the upper chambers before the invasion assessment. The bottom chamber was filled with 10% FBS-containing DMEM medium as the chemical attractant. The cells successfully migrating or invading the bottom chamber were stained with 0.1% crystal violet (catalog# C6158, Sigma-Aldrich, USA) and viewed under an optical microscope.

Dual-luciferase reporter assay

The wild-type and mutant-type ACTA2-AS1 vectors were constructed by cloning with predicted binding sites (on https://starbase.sysu.edu.cn/) or mutant binding sites into the pmirGLO vector (catalog# E133A, Promega, USA). To estimate the interaction between ACTA2-AS1 and miR-532-5p, the constructed vectors were co-transfected with miR-532-5p mimic, inhibitor, or NC into the MDA-MB-231 cell using Lipofectamine 2000 (Invitrogen, USA). The relative luciferase activity of ACTA2-AS1 was analyzed with the Dual-luciferase Reporter System (Promega, USA) and normalized to Renilla.

Statistical analyses

All data were expressed as mean value ± SD. according to triplicate experiments and determinations. The statistical analyses were carried out with SPSS 26.0 software. The differences between groups were evaluated with a student’s t-test or one-way ANOVA. The clinical data of patients were analyzed with the Chi-square test. The prognostic data proceeded with the Kaplan-Meier and multivariate Cox analysis. Meanwhile, the online Kaplan-Meier plot database (http://www.Kmplot.com) was also employed to further validate the prognostic significance of ACTA2-AS1 and miR-532-5p in breast cancer. The significant differences were represented by P < 0.05.

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