1.1 Cells and reagents
Human breast cancer cell lines BT-549, MDA-MB-231, MCF-7, and the normal human breast epithelial cell line (MCF10A) were purchased from the Kunming Cell Bank of Type Culture Conservation Committee of the Chinese Academy of Sciences.RPMI 1640 Medium, DMEM Medium, MEM Medium, MEpiCM Medium, and fetal calf serum were purchased from Gibco, USA. The siRNA was purchased from Biotechnology (Shanghai) Co., Ltd. The Lipofectamine RNAiMAX transfection and TRIzol reagents were procured from Invitrogen, USA. The real-time quantitative polymerase chain reaction (real-time fluorescence quantitative polymerase chain reaction, RTFQ-PCR) kit was purchased from Takara, Japan. The Cell Counting Kit-8 (cell counting kit-8, CCK-8) was purchased from Dojindo Corporation, Japan. Annexin V-fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) / propidium iodide (propidium iodide, PI) Apoptosis detection kit was purchased from Jiangsu Kaiki Biotechnology Co., Ltd.
1.2 Cell culture and passage
Human normal breast epithelial cell line MCF10A and human breast cancer cell lines BT-549, MDA-MB-231, and MCF-7 were added to MEpiCM Medium,RPMI 1640 Medium, DMEM Medium, and MEM Medium containing 10% fetal bovine serum, respectively, and cultured in an incubator with 5% CO2 at 37 ℃. After the cells reached 80-90% confluency, cells in good growth condition were subcultured .
1.3 Detection of LncRNA FOXD3-AS1 mRNA expression level
The total RNA of cells and tissues was extracted by the TRIzol reagent and reverse transcribed into cDNA using an RT-PCR kit as per the manufacturer’s instructions. With GAPDH as the internal control, the relative expression of LncRNA FOXD3-AS1 was determined and analyzed by the 2-ΔΔCT method. The primer sequence is as follows (Table 1):
Table 1 Sequences of primers used for RT-PCR
Primer name
|
Sequences (5’-3’)
|
FOXD3-AS1
|
F: GGT GGA GGA GGC GAG GAT G
R: AGC GGA CAG ACA GGG ATT GG
|
GAPDH
|
F: UGA CCU CAA CUA CAU GGU UTT
R: AAC CAU GUA GUU GAG GUC ATT
|
F: forward; R: reverse.
1.4 SiRNA down-regulated the expression of LncRNA FOXD3-AS1 in breast cancer cells 1.4.1 Small interference RNA transfection: According to the full-length LncRNA FOXD3-AS1 sequence, three small interference RNAs (FOXD3-AS1-Homo-135 (si1),FOXD3-AS1-Homo-560 (si2), and FOXD3-AS1-Homo-382 (si3)) were designed and synthesized by the Shanghai Jima Company, and three specific small interference RNAs (si1,si2 and si3) and small interference RNAs against GAPDH (siNC) were transfected using the Lipofectamine RNAiMAX transfection reagent. The sequence is as follows (Table 2):
Table 2 Sequences of siRNA
siRNA name
|
Sequences (5’-3’)
|
FOXD3-AS1-Homo-135 (si1)
|
S: GGA GCG GCU UUA AAG AGU ATT
A: UAC UCU UUA AAG CCG CUC CTT
|
FOXD3-AS1-Homo-560 (si2)
|
S: GUG GAC AAA UCC UCC AAG ATT
A: UCU UGG AGG AUU UGU CCA CTT
|
FOXD3-AS1-Homo-382 (si3)
|
S: GGC UAU UGA UUA AUC UAC UTT
A: AGU AGA UUA AUC AAU AGC CTT
|
small interference RNAs against GAPDH (siNC)
|
S: UUC UCC GAA CGU GUC ACG UTT
A: ACG UGA CAC GUU CGG AGA ATT
|
S: sense; A: antisence.
1.4.2 Detection of transfection efficiency: Human breast cancer cell lines (BT-549, MDA-MB-231, and MCF-7) were cultured in 6-well plates, with the cells evenly distributed. After 24 hours of incubation, the cells were transfected when a cell confluency of 60% was reached. 48 hours after transfection, Total RNA was isolated using the TRIzol reagent and real-time PCR was performed using an RT-qPCR kit to determine the relative expression of LncRNA FOXD3-AS1 so as to ascertain the transfection efficiency.
1.4.3 Cell proliferation ability:2×103 cells were plated in 96-well plates and transfected 24 hours later. After transfection, the cells were cultured for 0, 24, 48,72, and 96 hours, respectively. According to the CCK-8 manual, the absorbance (OD) of each well was measured, and the experiment was performed in triplicate.
1.4.4 Wound healing assay : The cells were seeded in 6-well plates and transfected with si-NC or si1 when a confluency of 70% was reached. Next, a 200 μL gun head from NEST Company was used to draw a line, cleaned with PBS, and a specific position was selected under the microscope as the 0h control. The corresponding positions were photographed again after 24 hours and 48 hours, respectively, and the change in the scratch area was measured.
1.4.5 Transwell migration assay: The transfected BT-549 and MDA-MB-231 cells were collected, and a 200 μL cell suspension containing 3×104 cells was added to the upper chamber of the Transwell chamber, while a 600 μL medium containing 20% fetal bovine serum was added to the lower chamber. After being cultured in an incubator at 37 ℃ for 24 hours, the unmigrated cells in the upper chamber were gently wiped with cotton swabs, fixed with methanol for 30 minutes, and then stained with Crystal violet dye (Biyuntian Biotechnology) for 15 minutes. The number of cells passing through the Transwell chamber was observed under a 10×10-fold microscope, and 4 visual fields were randomly selected, and images were taken. The experiment was repeated in triplicate, and the average number of cells was calculated and evaluated using graphs.
1.4.6 Transwell invasion assay: The Matrigel glue (Corning, USA) was diluted with a serum-free medium at 1:5. 100 μL of the solution was added to the upper chamber of Corning’s Transwell chamber and placed in a 37 ℃ incubator for solidification. Approximately 3×104 transfected BT-549 and MDA-MB-231 cells were suspended in a 100 μL serum-free medium and added to the upper chamber of the Transwell chamber. Afterward, a 600 μL medium containing 20% fetal bovine serum was added to the chamber. The method for observation and analysis was identical to that of the Transwell migration assay.
1.4.7 Detection of apoptotic ability: According to the manufacturer’s instructions, Annexin Ⅴ-FITC and PI were mixed with the transfected cells and detected by flow cytometry.
1.4.8 Western blot: The transfected cells were washed with PBS 3 times, then the RIPA lysate containing various protease inhibitors was added, and the cells were scraped off. After 20 minutes of lysis on ice, the solution was mixed, centrifuged at 4 ℃ and 12000 r/min for 20 minutes, and the supernatant was subsequently collected. After being quantified by the BCA protein quantitative kit (Biyuntian Biotechnology), the protein was mixed with 4 × loading buffer at a ratio of 1:3. The protein sample was heated for 10 minutes, and the sample volume was calculated according to the standard curve. The samples were separated by 8% polyacrylamide gel electrophoresis at a voltage of 60 V, followed by electrophoresis at an adjusted voltage of 90 V, and then transferred to PVDF membranes. Then, the membranes were blocked with 5% skimmed milk for 1 hour, washed with PBST 3 times, and incubated with the primary antibodies, namely, GAPHD and β-Catenin at 4 ℃ overnight. After washing three times with PBST, the membranes were incubated with the HRP secondary antibody for 1 hr at room temperature. Finally, the bands were developed using a chemiluminescence kit (Pierce, USA).
1.5 Statistical methods: Statistical analyses were performed by the SPSS 26.0 statistical software. All data were expressed as mean ±standard deviation. Comparison between two groups was determined by the t-test, and a p-value less than 0.05 was considered statistically significant.