Thirty paired BC tissues and adjacent normal breast tissues were procured from patients undergoing surgical excision at The First Hospital of Jilin University (Jilin, China). All patients did not receive any preoperative treatments for cancer. The collected tissue samples were promptly frozen with liquid nitrogen and preserved at -80℃ for the following experiments. Written informed consents were signed by all enrolled patients. This research was approved by the Ethics Committee of The First Hospital of Jilin University (Jilin, China).
BC cell lines (HCC1937, BT-20, MCF-7, and MDB-MB-436) and a human normal breast cell line (MCF-10A) were commercially obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% FBS at 37℃ in the presence of 5% CO2.
Particular shRNAs against NCK1-AS1 (sh-NCK1-AS1#1 and sh-NCK1-AS1#2) or SP1 (sh-SP1#1 and sh-SP1#2) and their corresponding negative controls (sh-NC) were acquired from GenePharma (Shanghai, China). In addition, GenePharma produced miR-361-5p mimics, NC mimics, and NC inhibitor. Above plasmids were respectively transfected into BT-20 or MCF-7 cells for 48 h. Cell transfection was conducted using Lipofectamine 2000 (Invitrogen, USA).
Trizol reagent (Invitrogen, USA) was utilized to isolate total RNA from tissues or cells according to the manufacturer’s instructions. PrimeScript™RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) was adopted to conduct reverse transcription. The expressions of NCK-AS1, miR-361-5p and SP1 were examined by RT-qPCR with SYBR Green (Takara Bio, Inc., Dalian, China). GAPDH functioned as an internal reference. The relative expressions were estimated by the 2-ΔΔCt method.
Sphere formation assay
BT-20 and MCF-7 CSCs (2×103/well) were incubated in ultra-low adhesion plates (Corning Incorporated, USA) with serum-free medium including 100 µg/mL human bFGF, 20 µg/mL insulin and 10 ng/mL human EGF. Two weeks later, the spheres were stained and counted by an inverted microscope (Nikon TE2000-U).
Western blot analysis
Total protein was extracted from cells utilizating a Total Protein Extraction kit (Nanjing KeyGen Biotech Co., Ltd., China). Protein BCA kit was conducted to measure the concentration of proteins. Then, the proteins were electrophoresed on SDS-PAGE and transferred onto PVDF membranes (Millipore). Afterwards, the membranes were sealed in 5% non-fat milk, and incubated with primary antibodies at 4℃ all night. The next day, appropriate secondary antibodies were added and incubated for 1 hour at room temperature (RT). Protein bands were visualized with ECL detection system (Thermo Fisher, Rockford, IL, USA). The GAPDH antibody was used as the internal reference. The primary antibodies were presented as follows: Sox-2 (ab97959); Oct-4 (ab19857); Nanog (ab80892); GAPDH (ab8245).
Cell counting kit-8 (CCK-8) assay
CCK-8 assay was performed to check cell viability in line with the supplier's instructions. Briefly, transfected BT-20 or MCF-7 cells were planted into 96-well plates (3×103 cells/well) and cultivated for 0, 24, 48 and 72 h. Each well was added with CCK-8 reagent and incubated for 2 h at 37℃. Finally, the absorbance (OD) at 450 nm was confirmed.
Colony formation assay
For colony formation assay, BT-20, or MCF-7 cells were harvested and planted into 6-well plates (1×103 cells/well). Two weeks later, the cells were immobilized, and dyed with 0.1% crystal violet. The colonies were counted by a gel documentation system (Bio-Rad).
Cell migratory and invasive abilities were examined by transwell assays. For invasion assay, BC cells (3×104 cells/well) were seeded in the upper chamber coated with Matrigel (Corning Incorporated). The lower chamber was added with 600 μl DMEM comprising 10% FBS. 48 hours later, the invaded cells were immobilized using ethanol, stained in crystal violet, and counted under a microscope. The migration assay was conducted the same steps without coating the membranes with Matrigel.
Fluorescence in situ hybridization (FISH) assay
FISH assay was applied to confirm the location of NCK1-AS1 in BT-20 or MCF-7 cells. The NCK1-AS1 probe was obtained from RiboBio, Co., Ltd. (Guangzhou, China). BT-20 or MCF-7 cells were cultured with NCK1-AS1 probe overnight at 37 ℃, and then blocked by 3% BSA. The nucleus was stained using DAPI. The fluorescence microscope (Olympus Optical Co., Ltd., Japan) was used to capture the images under 5 different visual fields.
RNA pull-down assay
GenePharma constructed NCK1-AS1 biotin probe and NCK1-AS1 no-biotin probe. Then the probes were cultivated with streptavidin magnetic beads (BioMag, Shanghai, China) for 1 h at RT. Cell lysates of BT-20 and MCF-7 cells were cultured with probe-coated beads at 4℃. Finally, the pulled-down RNAs were eluted and the expressions of microRNAs (miRNAs) were detected using RT-qPCR.
Luciferase reporter assay
To construct the pmirGLO-NCK1-AS1 WT/Mut and pmirGLO-SP1 WT/Mut vector, the sequences comprising the wild-type or mutant binding sites targeting miR-361-5p of NCK1-AS1 and SP1 were subcloned into the pmirGLO luciferase vector (Promega, USA). NCK1-AS1 WT/Mut vector or SP1 WT/Mut were transfected with miR-361-5p mimics, or respective controls (NC mimics) in BT-20 and MCF-7 cells. Moreover, the sequences containing NCK1-AS1 promoter region was cloned into the pGL3 luciferase vector (Promega, USA). The vector was respectively transfected into BT-20 and MCF-7 cells with sh-SP1#1, sh-SP1#2, or sh-NC. Luciferase activity was measured using the Luciferase Reporter Assay System (Promega, USA) 48 hours later.
RNA immunoprecipitation (RIP)
The EZ-Magna RIP kit (Millipore, USA) was used to conduct RIP assay. After centrifugation and collection, BT-20 and MCF-7 cells were lysed in RIP lysis buffer (Thermo Fisher Scientific). Antibody against Ago2 (Millipore) or negative control IgG (Millipore) was conjugated with magnetic beads. The enrichment in immunoprecipitation was detected finally.
Chromatin immunoprecipitation (ChIP)
The combination between SP1 and NCK1-AS1 promoter was examined using ChIP assay. The EZ ChIP Chromatin Immunoprecipitation Kit (Millipore, USA) was adopted for ChIP assay as per the manufacturer’s directions. Briefly speaking, anti-SP1 antibody (Millipore, USA) was applied to immunoprecipitate the chromatin, and IgG served as negative control. Finally, coprecipitated DNA was purified and the expressions of target genes were examined using RT-qPCR.
SPSS 20.0 Software (SPSS Inc., Chicago, IL) was used to analyze data. All results are displayed as the mean ± SD by at least three independent assays. Data were calculated under Student's t test (two groups) or one-way ANOVA (multiple groups). Statistical significance was considered when p-value was < 0.05.