Cell lines and transfection. MDA-MB-231 and BT-549 cells were obtained from the Chinese Academy of Sciences (Shanghai, China). The MDA-MB-231 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the BT-549 cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA); both media were supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and 100 IU/ml streptomycin (both from Thermo Fisher Scientific, Inc.) at 37℃ in a humidified atmosphere containing 5% CO2. The appropriate number of cells was seeded into 6-well plates, and cell growth confluence reached approximately 80% before transfection was performed with Lipofectamine 3000 (Invitrogen, L3000015) transfection reagent, according to the manufacturer’s instructions.
Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) assay. Total RNA was extracted from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). For the detection of miR‑1246 expression, a miRNA reverse transcription kit (Thermo Fisher Scientific, Inc.) was used to convert RNA into cDNA according to the manufacturer's instructions. qPCR was performed using a miRNA Q‑PCR detection kit (GeneCopoeia, Rockville, MD, USA) on an Applied Biosystems 7500 thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The final reaction volume was 20 µl, which included 1 µl of cDNA, 10 µl of PCR master mix (GeneCopoeia), 2 µl of primer and 7 µl of H2O. The PCR conditions were as follows: 95°C for 5 min and 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 30 sec. Primers with the following sequences were purchased from Sangon Biotech Co., Ltd. (Shanghai, China): miR‑1246 forward, 5'‑ TGAAGTAGGACTGGGCAGAGA ‑3' and reverse, 5'‑ TTTGGGTCAGGTGTCCACTC ‑3'; and U6 forward, 5'‑CGCTTCGGCAGCACATATAC‑3' and reverse, 5'‑AAATATGGAACGCTTCACGA‑3'. The U6 gene was used as an internal reference. The relative expression levels were analyzed using the 2‑ΔΔCq method, which was described in our previous publication (10).
Immunoprecipitation and immunoblotting. Cells were lysed with RIPA lysis buffer and extraction buffer. Then, the samples were precleared by incubation with 20 µl of protein G beads (Amer-sham Biosciences) for 2 hours, followed by incubation overnight with 1–2 µg of the anti-PGRN primary antibody (Abcam, ab108608) at 4°C. Protein G beads were then added to the sample tube and further incubated for 2 hours. The samples were then centrifuged at 10,000 g for 1 min at 4°C, and the pellets were washed three times with immunoprecipitation buffer. For immunoblot analysis, bound proteins were boiled at 100°C for 10 min in SDS-PAGE loading buffer, and then, a western blot assay was performed.
Western blot analysis. Total protein was extracted according to the manufacturer’s instructions of the protein extraction kit, and protein concentrations were measured via the BCA method. The proteins were subjected to 10% SDS-PAGE for electrophoresis and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 hours and then washed with TBST. The membrane was incubated overnight at 4°C with primary antibodies. The primary antibodies added included anti-DYRK1A (CST, 2271S), anti-N-cadherin (Abcam, ab124397), anti-E-cadherin (ICL, YT1454), anti-vimentin (ICL, YT5796), anti-p-GSK3β (Abcam, ab93926), anti-GSK3β (Abcam, ab131097), anti-β-catenin (Proteintech, 51067-2-AP), anti-PGRN (Abcam, ab108608), anti-p-GSK3β, anti-GAPDH (Goodhere Biological Technology, AB-P-R 001), and anti-lamin B (Boster, BA1228). After washing, the membranes were subsequently incubated with secondary antibody for 1 hour at 37°C. An ECL chemiluminescence kit was used to visualize the protein bands. GAPDH was used as the loading control as described in our previous publication (10).
Cell proliferation assay. We used a CCK-8 kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions to examine cell proliferation. Approximately 1×104 TNBC cells were seeded into 96-well culture plates. After 24, 48, or 72 hours of transfection, the cells were inoculated with 100 µL of WST-8 at 37°C for 4 hours. The absorbance of the cells was then measured at OD450.
Cell migration assay. TNBC cells were cultured in a 6-well plate at a density of 1×105 cells per well. After 24 hours in culture, the cells were transfected with Lipofectamine 3000. After 12 hours, wounds (1-mm wide) were created using a plastic scriber. The scratch wounds were photographed over 24- and 48-hour periods using a microscope (CX31; Olympus Corporation, Tokyo, Japan).
Transwell assay. According to the manufacturer's protocol, Transwell experiments were performed to evaluate cell migration and invasion abilities. Briefly, 1×105 TNBC cells were prepared in serum-free DMEM or RPMI-1640 medium and added to the upper chamber of a Transwell plate coated with Matrigel (BD Biosciences, NJ). Then, 500 µL of DMEM or RPMI-1640 medium with 10% FBS was added to the lower chamber containing MDA-MB-231 or BT-549 cells. After incubation for 24 hours, the cells were fixed using paraformaldehyde, stained with 0.1% crystal violet, and counted under a microscope (CX31; Olympus Corporation, Tokyo, Japan).
Luciferase reporter gene assay. Total cDNA from MDA-MB-231 cells was used to amplify the 3'UTR of DYRK1A using PCR, and the sequence was then cloned into a pmirGLO‑Report vector (Thermo Fisher Scientific, Inc.), resulting in the generation of pmirGLO‑DYRK1A. Mutations were introduced within the seed sequences of the 3'UTR of DYRK1A using a QuikChange site-directed mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA). The mutated DYRK1A 3'UTR was then cloned into the pmirGLO‑Report vector, generating pmirGLO-DYRK1A-Mut. 293T cells were cotransfected with 100 ng of the pmirGLO ‑DYRK1A or pmirGLO‑DYRK1A‑Mut vector and 100 mM miR‑1246 mimic or scramble miR mimic, and a pRL‑TK plasmid (Promega Corporation, Madison, WI, USA) was used for internal normalization. Following a 36-hour incubation at 37°C in an atmosphere containing 5% CO2, the cells were lysed using lysis buffer (Promega Corporation), and a dual-luciferase reporter assay system (Promega Corporation) was used to conduct a luciferase reporter gene assay on an LD400 luminometer (Beckman Coulter, Brea, CA, USA) according to the manufacturer's instructions. The data are presented as the ratio of Renilla luciferase to firefly luciferase.
Statistical analysis. GraphPad Prism 9 software (La Jolla, CA, USA) was used for statistical analysis. The statistical data are presented as the means ± standard deviation (SD). Student's t-test was used to compare groups, with p values < 0.05 considered to be statistically significant.