Design and preparation of dsRNA
saRNA targeting the promoter of p21 at position-322 relative to the transcription start site was termed as dsP21-322 and designed as previously described . Scramble dsRNA with the following sequence: S, 5'-UUCUCCGAACGUGUCACGU [dT][dT]-3'; AS, 5'-ACGUGACACGUUCGGAGAA[dT][dT]-3' was also synthesized and used as control. Synthetic dsRNAs were manufactured by Genepharma Inc (Shanghai, China).
Cell culture and transfection
Human lung carcinoma cells (A549) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum and penicillin (100 Units/ml)/streptomycin(0.1 mg/ml) in 5% CO2 incubator at 37°C. Cells were seeded into six-well plates with growth medium at a density of 0.8 × 105 cells/well respectively and cultured overnight to (30-50)% confluence prior to transfection. Cells were then transfected with 100 pmol/well of dsP21-322 or scramble dsRNA, respectively, using the LipofectamineTM2000 reagent (Invitrogen, USA) according to the manufacturer's protocols.
RNA isolation and semi-quantitative RT-PCR
Total RNAs were extracted from dsP21-322, scramble dsRNA and mock transfected A549 cells by using TRIzol reagent according to the manufacturer's instructions. Complementary DNA (cDNA) was generated from total RNA by reverse transcription using moloney murine leukemia virus (M-MLV). PCR amplification of the cDNA was performed in a reaction mixture with a final volume of 30 μL containing 2 μL of 4 × dNTPs, one unit of Taq DNA polymerase, and 10 mmol/L of each paired primer specific to p21 gene. The primers used for RT-PCR of p21 were forward primer, 5'-TTGATTAGCAGCGGAACA-3' and reverse primer, 5'-TACAGTCTAGGTGGAGAAACG-3'.
The cells from experiment group and control groups were harvested and washed with PBS (pH 7.4) twice and resuspended in lysis buffer (1 mM dithiothreitol, 0.125 mM EDTA, 5% glycerol, 1 mM phenylmethylsulfonylfluoride, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 1 μg/mL aprotinin, 1% Triton X-100 in 12.5 mM Tris-HCl buffer, pH 7.0) on ice. The cell extracts were clarified by centrifugation and the protein concentrations were determined by using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Each protein extract (25 μg) was electrophoresed on a 12% SDS-polyacrylamide gel, transferred to PVDF membrane in a buffer containing 25 mM Tris-HCl (pH 8.3), 192 mM glycine, 20% (v/v) methanol, and blocked in 5% (w/v) skimmed milk in Tris buffered saline-Tween 20 (0.1% by volume, TBST) for 1 hour at room temperature, and probed with specific primary antibodies overnight at 4°C. Then primary antibodies were removed and the blots were extensively washed with TBST for three times. Blots were then incubated for an hour at room temperature with the secondary antibodies (goat anti-rabbit/mouse antibody coupled to horseradish peroxidase, 1:3000 dilution) in 1% (w/v) skimmed milk dissolved in TBST. Following removal of the secondary antibody, blots were extensively washed as above for an hour and developed using the Enhanced Chemiluminescence Kit (NENTM Life Science Products Inc, Boston, MA). The primary antibodies used in this experiment for western blotting analysis were anti-p21 (1:100, Santa Cruz) and anti-β-actin (1:500, Sigma) antibody.
2.5 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2Htetrazolium bromide (MTT) assay
MTT assay was performed to assess the effect of p21 expression on cell proliferation. Transiently transfected lung carcinoma cells were plated in 96-well plate at a density of 3.0 × 103 cells/well for proliferation assay. Then for 5 days, every 24 h a batch of cells were stained with 20 μl sterile MTT dye (5 mg/ml; Sigma, USA) at 37°C for 4 h, then culture medium was removed and 150 μl of DMSO was added and thoroughly mixed in for 10 min. Spectrometric absorbance at 490 nm was measured by using a microplate reader. All experiments were performed in triplicate.
Colony formation assay
Approximately 0.5 × 103 A549 cells transiently transfected with dsP21-322, scramble dsRNA and mock were plated in 100-mm culture dishes, respectively. After 18 days, cells were fixed with methanol and stained with 0.1% crystal violet. Visible colonies were manually counted.
Flow cytometric analysis of apoptosis
An annexin V-fluorescein isothiocyanate apoptosis detection kit (Zymed, USA) was used to detect cell apoptosis. Approximately 1 × 106 A549 cells transiently transfected with dsP21-322, scramble dsRNA and mock, respectively, were harvested and analyzed by Flow Cytometry (BD, USA).
In vitro chemosensitivity assay
The dsP21-322, scramble dsRNA and mock transfected A549 cells were seeded in 96-well plate at a density of 5 × 104 cells/well. The cells were then treated with 5 μg/ml cisplatin for 48 h.Then, 20 μl of MTT stock solution (5 mg/ml) was added to each well, and the cells were incubated at 37°C for 4 h. The supernatant was replaced with DMSO to dissolve formazan production. The A490 nm values were assayed in a microplate reader. The ratio of the absorbance of treated cells relative to that of the control cells was calculated and expressed as a percentage of cell viability. The mean of three parallel samples was calculated. Experiments were performed in triplicate and standard deviations were calculated based on the average of three experiments.
In vivo chemosensitivity assay
A549 cells (1 × 106) were injected subcutaneously into the right posterior limb of BALB/c nude mice (4-6 weeks old). When palpable tumors (about 100-130 mm3) arose within 16-21 days, mice were randomized to treatment and control groups. Three groups (five mice each) received intratumoral injections of mixture of 30 μg of LipofectamineTM2000-encapsulated dsP21-322, scramble dsRNA and PBS respectively, every 3 days for 3 weeks. The other two groups received intratumoral injection of PBS combined with cisplatin or dsP21-322 combined with 5 mg/kg cisplatin, individually, every 3 days for 3 weeks. Tumor growth was monitored by caliper-measuring two perpendicular tumor diameters every 3 days, and the volume of the tumor was calculated from the formula: V = (width2 × length × 0.5). At the end of the experiment, tumor weight was assessed by sacrificing the mice, and by removing and weighing the tumor. Animal experiments in this study were carried out in accordance with the medicine institutional guidelines of Fourth Military Medical University.
Immunohistochemistry of tumors
The sections of the tumor tissues embedded in paraffin were stained using mouse anti-p21 antibody (Santa Cruz) at 1:50 dilution overnight at 4°C. After brief washing, all slides were stained and visualized with a Histofine SAB-PO(M) kit (Nichirei, Tokyo, Japan) according to the manufacturer's instructions.
Results were expressed as Means ± standard deviation (SD). Statistical analyses were performed using SPSS statistical software. Student's t-test and one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests were adopted. Values of p < 0.05 were considered as significant and indicated by asterisks in the figures.