Patients and sample collection
A total of 74 cases of initial diagnosis of DLBCL admitted to our hospital from February 2012 to November 2013 were enrolled in current study. No previous chemotherapy, radiation or other biological treatments were considered as the inclusion criteria. Meanwhile, the other types of lymphoma, and DLBCL combined with other diseases were considered as the exclusion criteria. Moreover, a total of 26 patients with pathological diagnosis of reactive lymphoid hyperplasia were selected as controls. The specimens were excised during surgery, and then stored in liquid nitrogen at 80°C until RNA extraction. The overall survival (OS) was defined as the date from enrollment to death. This study has been approved by the ethics committee of Shouguang People's Hospital of Shandong Province. All patients signed a written informed consent form prior to the study.
Human normal B cell immortalized cell line (HMy2.CIR), DLBCL cell line, central B cell-like (GCB) OCI-Ly19 and SU-DHL-4 cells, activated B cell-like ( ABC) OCI-LY10 and U2932 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HMy2.CIR was cultured in IMDM (Gibco, USA) medium containing 10% FBS and 1% penicillin-streptomycin (P/S). U2932 and SU-DHL-4 cells were cultured in RPMI 1640 medium (Gibco, USA) medium containing 10% FBS and 1% P/S. OCI-LY10 and OCI-Ly19 cells were cultured in IMDM (Gibco, USA) medium containing 20% FBS and 1% penicillin-streptomycin (P/S). All cells were cultured in a humidified humidity incubator with 5% CO2 at 37 °C.
Cell transfection and grouping
OCI-LY10 and U2932 cells in good growth condition were collected and seeded in 6-well plates (5×105 cells/well). miR-222-3p mimics, miR-222-3p inhibitors, mimics negative control (NC), inhibitors NC, pcDNA3.1-NC and pcDNA3.1-PPP2R2A (Shanghai Jima Company) (15 μl for each) were dissolved in 250 ml medium and mixed evenly to obtain A solution. Meanwhile, a total of 5 ml EntransterTM-R transfection reagent (Engreen Biosystem) was added to 250 ml culture medium and mixed evenly to obtain B liquid. Then, the solution A and B were mixed evenly and incubated in incubator for 48 h. According to different treatments, all cells were divided into miR-222-3p mimics group, mimics NC group, miR-222-3p inhibitors group and inhibitors NC group, mimics NC + pcDNA3.1-NC group, mimics + pcDNA3.1-NC group, mimics NC + pcDNA3.1-PPP2R2A group and mimics + pcDNA3.1-PPP2R2A group. The untransfected cells were named as Blank group.
The expression level of miR-222-3p was detected by qRT-PCR. Briefly, cells cultured for 48 h were collected to extract total RNA by using Trizol method, and reverse-transcribed using random hexamer primers (invitrogen, San Diego, USA) in accordance with manufacturers’ instructions. qRT-PCR was performed on ABI PRISMR 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The PCR program included 40 cycles of 95°C for 3 min, 95°C for 10 s and 55°C for 30 s. Data was analyzed by the 2-ΔΔCt method 17, and all oligonucleotide primers were designed and synthesized by Biotechnology Bioengineering Co., Ltd. (Shanghai). The primer sequences for samples and internal references were listed in Table 1.
Luciferase reporter gene assay
The target sites for miR-222-3p were determined by Target Scan (http://www.targetscan.org/), and the wild-type (PPP2R2A-WT) or mutant (PPP2R2A-MUT) of miR-222-3p were designed according to the predicted results. The miR-222-3p mutant sequence and the wild sequence fragment were cloned to the pGL3 luciferase control reporting vector (Promega, USA). Then, the EHK-293T cells (ATCC) were seeded into 24-well plates (5 × 105 cells/well) and co-transformed with PPP2R2A-WT (or PPP2R2A-MUT) and miR-222-3p mimic (or negative control) using Lipofectamine 2000 (Thermo Fisher Scientific). After transfection for 48 h, luciferase assay was determined by dual luciferase reporter assay kit (Promega).
RNA pull-down assay
miR-222-3p-Wt, miR-222-3p-Mut and miR-NC were transcribed using the TranscriptAid T7 High Yield Transcription Kit (ThermoFisher Scientific, Waltham, MA, USA). The Biotin RNA labeling cocktail (Roche Diagnostics, Indianapolis, IN, USA) was used to synthesize Bio-miR-222-3p-Wt, Bio miR-222-3p-Mut and Bio-miR-NC. Furthermore, a total of 50 pmol of biotinylated RNA was mixed with 200 μg of cell lysate (OCI-LY10 or U2932 cells), and then incubated with 50 μl of streptavidin agarose (Invitrogen, Carlsbad, CA, USA) for 1 hour at 4 °C. Fiannly, the eluted proteins were measured using RT-qPCR.
Transfected cells in the logarithmic growth phase were seeded in 96-well plates (6×103 cells per well), and cultured in incubator (37°C, 5% CO2) for 24-72h. MTT (5 mg/mL) was then added into each well with a volume of 20 μL. Culture was terminated after continuing incubation in the incubator for 4 h. Then, a total of 150 μl DMSO was added to each well to promote crystallization dissolution. The absorbance values of the wells at 0h, 24h, 48h and 72h were measured, followed by the construction of MTT plot (Y-axis: absorbance value; X-axis: interval time). The experiment was repeated for 3 times.
Clonal colony formation assay
Cells in each group were cultured for 48 hours after transfection, and then cells were washed with PBS and digested with 1% trypsin. All the cells were primarily added into 6-well plates (300 cells/well) with 2.5 mL medium in each well. Two weeks later, residual liquid in each well was discarded. After washed by PBS for twice, cells were fixed with 4 % paraformaldehyde solution and then stained with Swiss-Gimsa for 15 min. Then residual liquid in each well was discarded, all plates were dried at room temperature. Clones were counted automatically by using ImageJ (1.48V) software, and then photographed with an inverted phase contrast microscope (Olympus Ckx53). Cell colony formation rate was calculated as (number of colonies/total number of cells seeded) × 100%.
The effect of miR-222-3p mimics or inhibitors on OCI-LY10 and U2932 cells apoptosis was measured by Annexin V/FITC apoptosis detection kit (Kaiji Biotechnology Co., Ltd., China). Mixed solution consisting of 5 μL PI and 5 μL Annexin V/FITC was added to cells for 15 min incubation. Apoptosis was conducted by flow cytometry according to the procedure in the Annexin V-FITC/PI Assay Kit.
The logarithmic growth phase of OCI-LY10 and U2932 cells wer inoculated into the Transwell chamber (Corning, USA), and the cell density of each group was adjusted to 2×105/mL with serum-free RPMI-1640 medium. A total of 200 μL samples was added to the upper chamber, while 400 μL of RPMI-1640 medium containing 20% FBS was added to the lower chamber (24-well plate). After 48 hours of culturation, the chamber was removed and the medium in the plate was aspirated, washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with crystal violet for 20 min. Then room dried, and photographed under the microscope. Five random fields were used to count the number of transmembrane cells (mean and standard deviation) under a microscope (200 ×), and the number of cells passing through the cells was used to indicate the invasive ability.
Western blot analysis
Protein expression was measured by Western blot. Briefly, a total of 50 μg total protein was extracted by lysis buffer, followed by quantifying using a BCA kit (Nanjing Kaiji Biotech Co., Ltd.). Then samples was subjected to 10 % SDS-PAGE, and transferred onto PVDF membrane. Afterwards, the membrane was blocked with 5% skim milk in TBST solution. Subsequently, membrane was sequentially incubated with primary antibodies (Rabbit anti-human Bcl-2, Bax, PPP2R2A, 1:2000, abcam, UK) and the secondary antibodies (bs-0295G-HRP, 1:5000, Beijing Biosynthesis Biotechnology Co., Ltd., China). At last, blots were visualized by enhanced chemiluminescence Plus, and integrate optical density was measured by software Lab Works 4.5.
Tumor growth assay
A total of 18 SPF BALB/c nude mice (4-week-old) were purchased from SLACL laboratory animal center (Shanghai, China). Then, 0.1 ml of 1.0×107/ml OCI-LY10 cells from Blank, miR-222-3p mimics or mimics NC group, respectively were injected subcutaneously into the flank of nude mice (6 mice in each group). After transplantation, the tumor growth and tumor volume were observed every 7 days, and the final measurement was performed on the 56 th day. Tumor volume was estimated according to the following formula: L × W2/2 (where L represented the length, and W represented the width). At the end of the experiment (on day 56), mice were anesthetized (with CO2) and killed. Tumor tissue was dissected and collected for further analysis. All animal studies were approved by the Animal Ethics Committee of Shouguang People's Hospital of Shandong Province and were conducted in compliance with animal-use guidelines established in Shandong, China.
All statistical analyses were performed using SPSS 21.0 statistical software. The results were presented in the form of mean ± standard deviation (SD). The data of two groups were analyzed by the Student t test. P < 0.05 was considered to be statistically significant.
After the study, all animals were euthanized. The right hand held the rat tail and pull it back, and the left thumb and forefinger pressed down firmly on the mouse head at the same time. The external force was used to dislocate the cervical spine of the mouse, and the spine and the brain were disconnected. This method can quickly lose consciousness and reduce pain of experimental animals, which is a commonly used method for euthanasia of small experimental animals.