Cell Lines and Cell Culture
The HBZY-1 cell line was obtained from the Cell Bioscience Inc (Shanghai, China). After receiving the cells, the cells were placed in the incubator for 3 hours to stabilize the cell state. After washing with PBS (Hyclone, Logan, UT, USA), 1mL trypsin (Beyotime Biotechnology, Shanghai, China) was added for digestion for 2min. After centrifugation, the suspension was transferred to T-25 culture flask (KIRGEN, Shanghai, China) and cultured in high glucose Dulbecco's modified Eagle's medium (DMEM, Hyclone) containing 10% fetal bovine serum, penicillin (100 U/mL, Invitrogen, Carlsbad, CA, USA), and streptomycin (100 μg/mL, Invitrogen). The HBZY-1 cell line was cultured in a humidified incubator containing 5% CO2 at 37°C.
Cell transfection
HBZY-1 cells were divided into groups after synthesis of miR-339-5p inhibitors, miR-339-5p mimics and negative control (NC) sequences. The transient transfection sequence of Lipofectamine 2000 kit was used to transfect HBZY-1 cells, 24 hours after transfection, the gene expression of each group was detected, and the cells of each group were collected for cell cycle and slice climbing experiments. At the same time, the cells were collected and put in the refrigerator at -80℃ for follow-up Western blotting and qRT-PCR experiments.
Quantitative Real-Time Polymerase Reaction
Trizol reagent (Life Technologies, Carlsbad, CA, USA) was used to extract total RNA in each group. PrimeScript™RT Reagent Kit with gDNA Eraser Kit (TaKaRa, Kyoto, Japan) for reverse transcription into cDNA. SYBR qPCR SuperMix Plus (NovoProtein, Shanghai, China) is used for real-time quantitative polymerase chain reaction in fluorescent quantitative PCR apparatus (Thermo Fisher Scientific, Waltham, MA, USA). The reaction conditions were predenaturated for 1min at 95 °C in the first step. The second step is denaturation at 95 °C for 20s, annealing at 60 °C for 1min, 40 cycles. Using β-actin or U6 as control, the relative expression levels of related RNAs were calculated by 2-ΔΔCT method. Primer sequences of each indicator were shown in Tab. 1.
Western blotting assay
The cell samples were collected and the total cell proteins were extracted with RIPA lysis buffer (Beyotime), After electrophoretic separation and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Seal at room temperature for 2 hours in a Tris buffer containing 5% skim milk. The primary antibody was incubated overnight. The dilution ratio was Syk (1:500), p-Syk (1:1000), Ras (1:1000), MEK (1:1000), p-MEK (1:1000), ERK (1:1000), p-ERK (1:500) and c-Fos (1:500). After washing, secondary antibody (Abcam, Shanghai, Chnia) labeled with horseradish peroxidase was incubated at room temperature for 1h. Protein bands were obtained by automatic exposure apparatus (Shanghai Peiqing, China). The bands were quantified by Image J software (1.8.0).
Cell Counting Kit-8 Proliferation Assay
The HBZY-1 cells in the culture bottle were washed with aseptic PBS and digested with trypsin (Beyotime) for 2 min. After centrifugation, the cell count was adjusted to 5×104 cells/well, and inoculated in a 96-well plate, and 5% CO2 was incubated overnight in an incubator at 37 ℃. The cells of each group were transfected as required, and 10μL of CCK-8 solution (Bioss, Beijing, China) was added to each well of the 96-well plate. After 4 hours of culture, the absorbance of each hole was measured at the OD450 nm of enzyme-linked immunosorbent assay (Rayto, Shenzheng, China).
Immunofluorescence
The cells were inoculated in 6-well plates. 4% paraformaldehyde (Ebiogo, Anhui, China) was fixed for 20 min, goat serum (Zsbio, Beijing) was closed for 30 min, antibody was incubated for 60 min, the dilution ratio was Syk (1:300), p-Syk (1: 300), Ras (1:200), ERK1/2 (1:200), p-ERK1/2 (1: 200) and c-Fos (1:50). DAPI (Beyotime) was stained for 5 min and sealed with fluorescence quenching agent (Beyotime). Fluorescence microscope was used to observe and photograph was taken. Light repellent drip plus secondary antibody (1:400) (abcam), incubated at 37℃ for 30 min (Beyotime) and stained at room temperature for 5 min. The fluorescence intensity of each group was observed under fluorescence microscope (Motic,Xiamen,China).
Flow Cytometry Assay
The cells of each group were washed with aseptic PBS, digested with 1mL trypsin for 2 minutes, centrifuged with 1000rpm 5min, removed the supernatant, fixed with precooled anhydrous ethanol in the refrigerator at -20℃ for one hour, the fixed cells were washed twice with precooled PBS, added 20uLRNase, resuscitated cells, digested 30min in 37℃ water baths, then added PI staining solution 400ul resuscitated cells, stained 40min at 4℃. Flow cytometry (BECKMAN, Brea, CA, USA) was used to observe the changes of cell cycle in each group.
Luciferase Reporter Assay
The 3'-UTR mutation sequence and 3'-UTR wild sequence of Syk were inserted into pSI-Check2 (Hanbio Biotechnology, Shanghai, China) luciferase reporter vector respectively. The vectors containing Syk mutant and wild sequence were named Syk- mutant (Syk-mut) and Syk-wild type (Syk-wt). Syk-mut and Syk-wt, together with miR-399-5p mimic or miR-NC, were co-transfected into 293T cells by Opti-MEM low-serum medium and liposome 2000, respectively. The cells were collected 48 hours after transfection, and the relative luciferase activity was detected by double luciferase analysis kit (Promega, Beijing, China).
Statistical analysis
All experiments were repeated three times and data were presented as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS 19.0 statistical software, and the figures were drawn by GraphPad Prism 8.0. Aoneway analysis of variance (ANOVA) was used to calculate the P-values, which < 0.05 was considered to be statistically significant.