2.1 Clinical tissue collection
A total of 60 tissues, including 15 tissues in stage I + II and 15 tissues in stage III + IV, and 30 corresponding adjacent tissues were collected from Affiliated Hospital of Nantong University. All patients did not receive any chemotherapy and signed written informed consent. Moreover, the experimental procedures were approved by the Ethics Committee of Affiliated Hospital of Nantong University.
2.2 Cell lines
Breast cancer cell lines, including Hs-578T, MCF-7, ZR-75-30 and HCC1937, and human breast epithelial cell line MCF-10A were obtained from the Chinese Academy of Science (Shanghai, China). Cells were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM, KeyGEN, Nanjing, China) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Sigma-Aldrich, USA) in a humidified atmosphere with 5% CO2 at 37oC.
2.3 Cell transfection
Short hairpin RNA (sh)-MIR4435-2HG, sh-TMEM9B, miR-22-3p mimic, miR-22-3p inhibitor and corresponding negative control (NC) vectors were obtained from Gene Pharma Company (Shanghai, China). Lipofectamine 3000 (Invitrogen, USA) was performed for cell transfection based on the specification.
2.4 Cell Counting Kit-8 (CCK-8) assay
Transfected MCF-7 and ZR-75-30 cells at the density of 1 × 104 cells/well were maintained in 96-well plates and cultured for different time points (0, 24, 48 and 72 h, respectively). Then, cell viability was determined using a CCK-8 kit (Beyotime, Shanghai, China) according to the protocols of the manufacturer. The optical density was measured by the OD value at 450 nm by using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA).
2.5 Colony formation assay
Transfected MCF-7 and ZR-75-30 cells (1 × 103 cells/well) were seeded in six-well plates. The media was replaced with a fresh culture medium every 2–3 days for 2 weeks. Subsequently, MCF-7 and ZR-75-30 cells were stained with 10% crystal violet for 30 min and observed by using a microscope (Olympus, Tokyo, Japan).
2.6 Wound healing assay
Transfected MCF-7 and ZR-75-30 cells (5 × 105 cells/well) were seeded into a six-well plate. Once the cells were 100% confluent, a wound was made on the surface of the cell using a 200-µL tip, and a serum-free medium was used. After 0 and 48 h, MCF-7 and ZR-75-30 cells were observed under an inverted microscope (Tokyo, Japan), and the distance between the wounds was recorded.
2.7 Transwell migration and invasion assays
For migration and invasion assays, transfected MCF-7 and ZR-75-30 cells (1 × 106 cells/well) were seeded in the transwell chambers containing an 8 µm size porous membrane (Corning, NY, USA). The upper chamber was inserted without or with matrigel, while the lower chamber was added by 20% FBS. After 48 h incubation at 37oC, the non-migrating or invading cells in the upper chamber were removed by cotton wool. The migrated or invaded cells in the upper chamber were stained with 0.1% crystal violet solution (Sangon Biotech, Shanghai, China) and counted using a microscope (Olympus, Tokyo, Japan).
2.8 RNA extraction and qRT-PCR analysis
RNA from clinical tissues and cell lines was extracted according to the instructions of the TRIzol reagent, and the OD260/OD280 of the RNA solution was also detected. The reverse transcription reaction was conducted according to the instructions of the cDNA synthesis kit, and the PCR reaction solution was configured according to the instructions of the Taq enzyme mixture. The amplification program was set up for primer sequence as listed below, and a PCR amplification reaction was conducted. PCR reaction conditions: 50oC for 2 min, 95oC for 10 min (1 cycle), 95oC for 5 s, 65oC for 1 min, 75oC for 20 s (45 cycles); and 75oC for 5 min (1 cycle). Each real-time PCR was repeated 3 times. MIR4435-2HG forward, 5’-CGGAGCATGGAACTCGACA-3’, and reverse, 5’-CAAGTC TCACACATCCGGG-3’. MiR-22-3p forward, 5’-AAGCTGCCAGTTGAAGAACTGTA-3’, and reverse, 5’-GCTGTCAACGATACGCTACGTAAC-3’. TMEM9B forward, 5’-AAAGTCCGCCATTT TGCCAC-3’, and reverse, 5’-ATTCGGGGCTCTGTAGTCCT-3’. U6 forward, 5’-CTCGCTTCGGCA GCACA-3’, and reverse, 5’-AACGCTTCACGAATTTGCGT-3’. β-actin forward, 5’-TCGTGGAAGG ACTCATGACC-3’, and reverse, 5’-ATGATGTTCTGGAGAGCCCC-3’. The fold change in gene expression was calculated using the 2−ΔΔCT method after normalizing to the expression level of U6 and β-actin.
2.9 Western blot assay
Protein from breast cancer cell lines was isolated using RIPA lysis buffer and quantified with a BCA kit (Beyotime, Shanghai, China). Protein was separated via 12% SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore, USA). Subsequently, the membranes were blocked with 5% skimmed milk and treated with primary antibodies overnight at 4oC. Membranes were washed and probed with HRP-conjugated secondary antibody (1: 2, 000, ab6728, Abcam, USA) for 1 h at room temperature. Protein bands were visualized by enhanced chemiluminescence (EMD Millipore) and quantified using ImageJ software (version 4.3; National Institutes of Health). The primary antibodies used (all from Abcam) were as follows: Anti-E-cadherin (1: 1, 000, ab1416), anti-vimentin (1: 1, 000, ab92547), anti-α-smooth muscle actin (SMA, 1: 1, 000, ab32575), anti-TMEM9B (1: 1, 000, ab189253) and anti-β-actin (1: 1, 000, ab8227).
2.10 Immunofluorescence assay
Transfected MCF-7 and ZR-75-30 cells were cultured in six-well plates at a density of 1 × 105 cells/well. After fixing in 4% paraformaldehyde and permeabilization with 0.2% Triton X-100 in PBS, cells were treated with primary antibodies against α-SMA (1: 1, 000, ab32575) overnight at 4oC. Subsequently, cells were incubated with goat anti-rabbit IgG H&L secondary antibody (1: 1, 000, ab150077). The nuclei were counter-stained with DAPI. Positive staining was observed under a fluorescence microscope (Zeiss AG, Germany).
2.11 Subcellular fractionation analysis
Cytoplasmic and nuclear fractions were extracted from MCF-7 and ZR-75-30 cells using the NE-PER Nuclear and Cytoplasmic Extraction reagent (Thermo Fisher Scientific, Inc. USA). RNA from each fraction was measured by qRT-PCR. U6 was used as internal references for MIR4435-2HG in cytoplasmic and nuclear fractions, respectively.
2.12 RNA pull-down analysis
MCF-7 and ZR-75-30 cells were incubated with biotin-labeled miR-22-3p-wild-type (WT) or -mutant (Mut) for 48 h and lysed in the specific buffer. The lysate was incubated with magnetic beads and washed three times with precooled lysis buffer and salt buffer solution. Finally, the bound RNA was purified with TRIzol®. MIR4435-2HG or miR-22-3p enrichment was analyzed using qRT-PCR.
2.13 Dual-luciferase reporter assay
The sequence of MIR4435-2HG-WT and Transmembrane protein 9 domain family member B (TMEM9B)-WT and MIR4435-2HG-Mut and TMEM9B-Mut were inserted into the pmirGLO reporter vector (GenScript, Shanghai, China). Subsequently, pmirGLO-MIR4435-2HG-WT/Mut and pmirGLO-TMEM9B-WT/Mut were treated with miR-22-3p mimic or NC into MCF-7 and ZR-75-30 cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc., USA). Luciferase activity was measured using a dual-luciferase reporter assay system (Promega Corporation, USA).
2.14 Statistical analysis
GraphPad Prism 6.0 software was used for graphing. All data were expressed as mean ± standard deviation (SD). SPSS 17.0 software was used for statistical analysis. Data comparison between groups was performed by analysis of variance. A pairwise comparison between means was performed by t-test. P < 0.05 was considered statistically significant.