Cell lines and human BC specimens
Human BC cell lines (MCF-7, MDA-MB-468 and MDA-MB-231) and the immortalized normal breast cell line MCF-10A were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Indicated cells were cultured in DMEM containing 10% FBS (Thermo Fisher Scientific) was supplied at 37°C in a humidified atmosphere of 5% CO2. 60 BC samples were obtained from the Department of General Surgery, the Second Affiliated Hospital of Harbin Medical University. Fresh BC tissues and paired adjacent normal tissues were frozen in liquid nitrogen immediately after surgical excision and saved at -80°C. This research was approved by the Ethic Committee of Harbin Medical University.
qRT-PCR assay
To isolate total-RNA, the TRIzol reagent (Life Technologies, Carlsbad, CA, USA) was performed. For cDNA synthesis, the total RNA was retrotranscribed with the PrimeScript® RT Reagent Kit (Takara). The SYBR Premix Ex Taq™ Kit (Takara, Tokyo, Japan) was utilized to carry out qPCR. And RT-PCR was conducted on a 7500 RealTime PCR System. GAPDH was measured as the internal control. The sequences of the primers that were used as follows: for MBNL1-AS1: 5′- CTCCCGCTTCTTCTACCGAC -3′ (forward), 5′- TTGGTGCATTTTAAGGCGGC -3′ (reverse); for CENPA: 5′- GATTCTGCGATGCTGTCTG -3′ (forward), 5′- GCCTTTGGAACGGTGTT -3′(reverse); for ZFP36: 5′- TCCACAACCCTAGCGAAGAC -3’ (forward), 5′- GAGAAGGCAGAGGGTGACAG -3’ (reverse); for GAPDH, 5′- CTCCTCCACCTTTGACGCTG -3′ (forward), 5′- TCCTCTTGTGCTCTTGCTGG - 3′ (reverse). All data were calculated by 2−ΔΔCT method.
Cell transfection
In order to assess the MBNL1-AS1 overexpression vector, the lentiviral vector-MBNL1-AS1 and lentiviral vector -convirus (Genepharma, Shanghai, China) were transfected in cells. sh-MBNL1-AS1#1,sh-MBNL1-AS1#2 and sh-control were obtained from GenScript (Nanjing, China). The qRT-PCR assays were used to detect the expression of MBNL1-AS1. The sequences of the MBNL1-AS1 targeting shRNAs were: for MBNL1-AS1-shRNA#1: 5′- GATCCGAACGAAAGGAGCAGGGTATTTCAAGAGAATACCCTGCTCCTTTCGTTTTTTTA-3′ (sense), 5′- AGCTTAAAAAAACGAAAGGAGCAGGGTATTCTCTTGAAATACCCTGCTCCTTTCGTTCG-3′ (anti-sense); for MBNL1-AS1-shRNA#2: 5′- GATCCGCCAGAACCTAGTCTCATGTTTCAAGAGAACATGAGACTAGGTTCTGGTTTTTA-3’ (sense), 5′- AGCTTAAAAACCAGAACCTAGTCTCATGTTCTCTTGAAACATGAGACTAGGTTCTGGCG-3′ (anti-sense); for NC-shRNA: 5′- GATCCCCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTT -3′ (sense), 5′- AGCTAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAAGGG -3′ (anti-sense). siRNA, si-ZFP36, si-NC were obtained from RiboBio (Shanghai, China). Cells were transfected using the Lipofectamine 2000 (Invitrogen).
MTT study
Transfected cells were seeded in a 96-well plate whichhad 3 × 103 cells/well. Each well was added with 10 μL MTT (5 mg/mL) for different periods of time. After 4 hours of incubation, 150μl of dimethyl sulfoxide was added to dissolve the precipitates.The absorbance was then measured by detection at 560 nm using a microplate reader. The experiment was performed independently in triplicate.
Colony formation analysis
The BC cells were harvested and plated in a 6-well plate (0.2 × 103 cells/well) and cultured in incubators. After 14 days of culture, the plate was washed 3 times with PBS. The indicated BC cells were dried at 37°C for 15 minutes. Then the cell colonies formed was numbered under a digital camera.
Sphere formation assay
A total of 5 × 103 cells were plated in 6-well plates (Corning Life Science). EGF (20 ng/ml, Invitrogen), bFGF (10 ng/ml, Invitrogen), and 2% B27 (Invitrogen, Carlsbad, CA) was formulated in a medium. After 2 weeks, the number of spheroids was counted manually by a microscope.
Flow cytometry assay
Using flow cytometry with the use of propidium iodide (Sigma‐Aldrich, MO China) staining (5 μg/ ml), cell cycle analysis was performed after transfection. Then, the indicated cells were added with chilled ethanol and saved at 4 °C overnight. Then, the BC cells were centrifuged and resuspended in PBS and appended with 100μL RNase at room temperature. After 30 min, 100μl PI was appended, cells were incubated at 37°C in dark for 60 minutes. Cell cycle was determined by FACScaliber Flow Cytometer.
Western blot
Total protein from tumor lysateby a lysis buffer (Beyotime, Jiangsu, China) and separated using 10% SDS-PAGE. A PVDF membrane was transferred by the indicated protein, which was blocked with 5% non-fat dry milk. Primary antibodies, namely, Ab against ZFP36, Ab against CENPA, and Ab against GAPDH was incubated with the membrane. Subsequently, the secondary antibodies were incubated with the membrane for 1 h. After developed by enhanced chemiluminescent and exposure, immunoreactive protein band intensities were analyzed by Image software.
RNA pull-down
The biotin-labeled MBNL1-AS1 plasmid and biotin-labeled antisense RNA plasmid were respectively transfected in BC cells. Biotin-labeled RNA was bound to the Streptavidin agarose Beads, next mixed with indicated cell lysates. Indicated bound RNAs were isolated from the beads by washed and boiled for normal western blot. The assays were experimented in RNase-free conditions.
RNA immunoprecipitation (RIP) assay
RIP was performed with the use of the Magna RIP kit (Millipore, USA). The spectrophotometer (Thermo Scientific, USA) was used to examine the RNA concentration, and the bio-analyzer (Agilent, USA) was utilized to detect the RNA quality. The input control was the total RNA. Following, qRT-PCR analysis was used to test the results in order to demonstrate the bound targets.
Magnetic Luminex® performance assay
Magnetic Luminex® performance assay was performed based on our previous study(14). Briefly, NC or MBNL1-AS1 were transfected in MDA-MB-231 cells, which were incubated for 1 day. Following centrifugation, the supernatant was collected. Then the following human cytokines: PLK1, PAF, CENPA, YB-1, TWIST, YY1, KLF4, CUG2, E2F8, SALL4, RAE1 and PTPA were analyzed by FlexMAP 3D (Luminex®) platform.
Fluorescence in situ hybridization (FISH) assay
The MBNL1-AS1 subcellular localization was assessed by a FISH kit was obtained from Guangzhou RiboBio Co., Ltd. The 4% paraformaldehyde‐fixed cells were fixed in PBS. After washed by phosphate buffered solution with tween for three times, the anti-fluorescence quencher sealed the indicated cells, the images were taken by the FV1000 laser microscope (Olympus, Japan).
mRNA decay assay
Stable cells were added with 5 μg/mL actinomycin D. Following, qRT-PCR was performed to determine the CENPA mRNA. The data was performed independently in triplicate.
Animal studies
Animal studies were ratified by ethics committee of Harbin Medical University (Harbin, China). The BC cells were injected in the flank of four-week-old female nude mice (each group has 3 mice). The volume of tumor was recorded every week. Euthanasia of mice by overdose of pentobarbital sodium(200 mg/kg, intraperitoneally) and tumors were measured after 4 weeks.
Statistical analysis
All analysis were experimented three times at least. The measurement data in this study were exhibited as means ± SD. Student’s t-test was applied to compare the numeric variables between two groups. R software package version 3.0.0 and Graphpad Prism 5 were used. A p-value<0.05 was regarded as significant, P-value < 0.01 was very significant.