1.1 Cell culture
1.2 shRNA stable transfection
The target sequence of the MMP-9 shRNA is 5'-GCCTGCAACGTGAACATCTTCGACGCCAT-3' (GenePharma Co., Ltd, Shanghai, China). One day before transfection, the cells were digested with trypsin and then inoculated into a new sterile cell culture dish (the specification of the dish was selected according to the specific experimental requirements). The next day, cell density was observed (preferably around 80%) and appropriate amount of shRNA was transfected. Then the cells were cultured in a constant temperature incubator at 37℃ and 5% CO2 for 24 h, and then cultivated in a complete medium containing the eukaryotic screening drug G418 (Invitrogen, USA). The fresh medium was replaced every other day. After a week or so, when a large number of untransfected shRNA cells died, and monoclonal began to appear in successfully transfected cells, the screening drug concentration was halved and culture continued. Cell lines stably expressing exogenous genes can be obtained in about 2-3 weeks.
1.3 Real-time PCR
Total RNA of cells was extracted by Trizol (Invitrogen, USA) method. Reverse transcription was performed according to the instructions of the TIANGEN Kit (QuantScript RT Kit, KR103), and Real-time PCR was carried out by referring to the Thermo Kit instructions (PowerUp™ SYBR™ Green Master Mix, A25780). See Table 1 for the primer sequences of target gene and internal reference gene
The amplification conditions was as follows: pre-denaturation at 95 °C for 10 minutes; denaturation at 95 °C for 10 seconds; annealing at 2 °C for 20 seconds; extension at 72 °C for 10 seconds. In order to verify the specificity of the amplification product, a solution curve was added after PCR. The solution curve had a single peak for specific amplification, and a bimodal or multi-peak for non-specific amplification. All tests were performed on 96-well plates, with three duplicate wells for each sample, and internal parameters for detection of β-actin were set for relative quantification by 2-DDCT method.
1.4 Western Blot
Cells were lysed with a cell lysate to prepare a cell lysate. Protein concentration was determined using a BCA kit (KeyGen Biotechnology, Co., Ltd., Jiangsu, China, KGP902). The extracted protein was sampled at about 50 μg per well, and was separated by 10% SDS-PAGE gel electrophoresis before transferring to PVDF membrane. Next, the PVDF membrane was sealed with TBST buffer containing 5% skim milk powder at room temperature for 1h, and incubated with primary antibody in a refrigerator at 4℃ overnight. After cleaning with TBST for 3 times, the membrane was incubated at room temperature for 1h with the secondary antibody, which was then washed away by TBST and finally the membrane was displayed by ECL luminescence liquefaction. See Table 2 for the name, item number and working concentration of the antibody used in the experiment.
Image J software was employed to analyze the gray value of the target protein, and β-actin was set as the internal reference for statistical analysis.
1.5 Wound-healing assay
Before seeding the cells, three horizontal lines with the same spacing was drawn with a pen at the bottom of the 6-well plates, running through each well of the plate. Approximately 1×106 cells were seeded in each well, and the degree of cell confluence should be 100% after overnight. The next day, a micropipette tip was applied to create a wound in the direction of a ruler, perpendicular to the horizontal line at the bottom, and the gun tip should be vertical. The cells were then rinsed with PBS and added to fresh complete medium. After that, the well plate was cultured in a constant temperature incubator of 37 ℃ and 5%CO2. The culture plate was taken out At 0 hours, 6 hours, 12 hours and 24 hours, at which time points, pictures were taken under the microscope at the same visual field. The mean distance between cells was calculated by Image J software.
1.6 Cell invasion experiment
Diluted with double medium-free, 50 μg/ml Matrigel gel (BD, USA) was taken to prepare base adhesive. After adding 100 μl base adhesive to each well in the Insert chamber (Corning, USA) (8 μm), the chamber was placed in a 24-well plate and dried at 37 °C to form an artificial basement membrane. Before inoculating the cells, the prepared basement membrane was incubated with 100 μl/well double non-medium at room temperature for 40 minutes for hydration treatment. Each chamber was inoculated with 20000 cells. According to the needs of the experiment, 200 μl cell suspension was inoculated in each well, and the medium containing 1% fetal bovine serum was applied in the chamber, while 600 μl complete medium containing 10% fetal bovine serum was added to the 24-well plate in the lower chamber. After inoculation, it was cultured in a 5%CO2 incubator at 37 ℃ for 48 hours, followed by the removal of the medium and a triple meticulous rinse of the chamber with PBS at 100 μl/well. Then the 24-well plate was fixed with 600 μl of 4% paraformaldehyde at room temperature for 30 minutes, washed with PBS solution for 3 times, and then stained with 600 μl crystal violet per well for 5 minutes. After cleaning and drying, photos were taken at randomly selected visual field with a microscope, and at least 3 pictures of each well were obtained for statistical analysis.
1.7 Statistical analysis
All the collected data were processed by GraphPad Prims 6.0 software, and the statistical data was expressed as mean±standard deviation. Inter-group comparison of means was conducted by the t-test, while multi-group comparison of means was performed by analysis of variance (ANVOA). P<0.05 was considered to be statistically significant.