TNBC samples and corresponding adjacent samples were collected from Fifty-two TNBC patients at China-Japan Union Hospital of Jilin University（Jilin, China). Informed consents were signed by each patient. Prior to operation, no patients suffered radiotherapy or chemotherapy. After collecting, each sample was instantly preserved at -80°C in liquid nitrogen for further use. The use of tissues was obtained the approval from the Ethical Review Board of China-Japan Union Hospital of Jilin University（Jilin, China).
Human normal breast epithelial cell (MCF-10A), human TNBC cell lines (MDA-MB-468, HCC1937, MDA-MB-436, MDA-MB-231) and HEK293T cell line were all commercially provided by ATCC, and maintained in an incubator containing 5% CO2 at 37°C. For cell culture, DMEM medium (Thermo Fisher Scientific, CA, USA) supplementing 10% FBS and 1% Pen/Strep solution was applied.
The specific shRNAs targeting LINC00514 (sh-LINC00514#1/2) were obtained from GenePharma Company to silence LINC00514 via utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) with sh-NC as control. The pcDNA3.1/LINC00514, pcDNA3.1/CCDC71L and empty NC vector, and miR-6504-5p/miR-3139 mimics/inhibitor and NC mimics/inhibitor, were all provided by GenePharma Company. Cell samples were reaped after 48 h.
The extraction of total RNAs from tissues and cells was conducted by TRIzol reagent (Invitrogen) with a RecoverAll™ Total Nucleic Acid Isolation kit (Ambion). Utilizing the Prime Script™ RT reagent kit (Takara, Dalian, China), reverse transcription reactions were performed. Later, the employment of SYBR Premix Ex Taq (Takara Bio, Shiga, Japan) was for RT-qPCR on StepOnePlus System (Applied Biosystems). Based on 2-ΔΔCT method, gene expressions were calculated with normalization to GAPDH or U6.
Colony formation assay
Transfected TNBC cells were planted to 6-well plates, and each well was filled with 5 × 103 cells. After two weeks, cells were fixed by 5% paraformaldehyde, and then 0.1% crystal violet solution was supplemented for staining. Finally, colonies (more than 50 cells) were counted and recorded.
For the measurement of cell proliferation, Edu assay kit (RiboBio, China) was used. In brief, TNBC cells in each group with treatment of EdU were stained by DAPI. Under fluorescence microscope (Nikon, Japan), visualized images of EdU-positive cells were obtained.
Flow cytometry analysis
TNBC cells were planted in 6-well plates and then rinsed in PBS. Later, 1 μL of PI (Invitrogen) and 2.5 μL Annexin V conjugated with FITC were added into binding buffer. Subsequently, the binding buffer was used to resuspend the cells after trypsinization. After 15 minutes, apoptotic TNBC cells were identified by flow cytometry (BD Biosciences).
Western blot analysis
By RIPA buffer (Thermo Fisher Scientific), proteins of TNBC cells were lysed, and then protein concentration was confirmed by BCA-kit (Beyotime, Shanghai, China). Separated by SDS-PAGE, proteins were transferred to PVDF membranes. Then, the membranes were blocked in 5% skim milk and incubated with primary antibodies (Abcam, Cambridge, MA) overnight at 4 °C. Washed by 0.1% TBST in triple, the membranes were incubated with secondary antibody at 37 °C for an hour. The loading control was GAPDH. The results were analyzed and visualized by ECL detection reagent (GE Healthcare, Chicago, IL).
Wound healing assay
To make cells adhere, TNBC cells were cultured in 96-well plates all night with each well filled by 5 × 104 cells. Later, sterile pipette tip was used to scratch the wounds. After 24 h, wounds were imaged following washing in PBS.
Transwell invasion assay
Transfected TNBC cells were reaped and placed into the upper transwell chamber (8 μm pores) coated with Matrigel (BD Biosciences). The serum-free medium was placed in the upper chambers, while the lower chamber was supplemented with medium containing 10% FBS. Incubated for 24 h, cells were fixed and stained. Then, an inverted microscope was used for the counting of invasive cells.
Subcellular fractionation assay
PARIS Kit (Invitrogen) was used to isolated nuclear and cytoplasmic RNA in TNBC cells. Then, the extraction of subcellular fractions was carried out. Later, the fractions were subjected to RT-qPCR with normalization to GAPDH (cytoplasm control) or U6 (nucleus control).
RNA pull down assay
LINC00514 and NC-lncRNA labeled with biotin were transfected into MDA-MB-468 and HCC1937 cells. The lysates of TNBC cells were used for conducting an incubation with streptavidin magnetic beads for 4 h at 4 °C. Subsequently, precooled lysis buffer and salt buffer was applied to rinse the beads. With the extraction of pull-down RNAs, the levels of miRNAs binding to LINC00514 were detected.
Luciferase reporter assay
LINC00514-WT/Mut and CCDC71L-WT/Mut vectors were separately constructed by cloning wild type (WT) and mutant (Mut) miR-6504-5p or miR-3139 binding site in LINC00514 sequence or CCDC71L 3′-UTR to pmirGLO (Promega) vectors. Then, above luciferase vectors were transfected with miR-6504-5p mimics, miR-3139 mimics or NC mimics into TNBC cells for 48 h. Finally, luciferase activity was examined with Dual Luciferase Assay System (Promega).
Through using GraphPad Prism 6 (GraphPad), data comparison between or over two groups were statistically analyzed by Student's t test or one-way ANOVA. Three biological repeats were included in all experimental procedures with results presenting as mean ± SD. The analysis of correlation between genes was conducted by Spearman's correlation analysis. P less than 0.05 was considered as cut-off value.