Construction and identification of miR-24 high expression plasmid
The miR-24 sequence was obtained from the http://www.miRbase.org website, which was 5’-UGGCUCAGUUCAGCAGGAACAG-3’. The anti-miRNA-24 was used to down-regulate miR-24 and interfere with its action, and the sequence was complementary to miR-24, i.e., 5’-ACCGAGUCAAGUCGUCCUUGUC-3’. The control sequence was as follows: 5’-GUCAUCAGUCGAGCUAGACGAG-3’. First-strand DNA was obtained, as well as the corresponding double-stranded DNA. After amplification through PCR, the in vitro recombination was performed, and the fragments were inserted into the pEGP-miR vectors (Cell Biolabs, San Diego, CA, USA). The following vectors were obtained: the pEGP-miRNA-24, pEGP-anti-miRNA-24, and pEGP-control vectors. These vectors were transformed into competent E.coli DH5α host strains, which were cultured on the ampicillin-resistant plates. The correct monoclonal colonies were picked, and after shaking, the plasmid DNA was obtained and subjected to the DNA sequencing (Sangon Biotech, Shanghai, China).
HUVEC culture and transfection
HUVECs were provided from Procell, Wuhan, Hubei, China. These cells were cultured with the 1640 medium (Gibco, Mt Waverley, Australia), containing 1 × 105 U/L penicillin and 100 mg/L streptomycin, and 10% fetal bovine serum (FBS; Gibco), in a 37 °C, 5% CO2 humidified incubator. For the cell transfection, the extracted plasmid concentrations were measured using a NANO nucleic acid analyzer. Then the plasmid was diluted with serum-free 1640 medium to the concentration of 0.01 µg/µL, which was mixed with the X-treme GENE HP DNA transfection agent (with the ratio of 1 µg: 3 µL; Roche Diagnostics, Mannheim, Germany). The normal HUVECs were cultured with the mixture medium for 24 h, which was replaced by the 1640 medium containing 10% FBS, and the fluorescence was detected. These cells were divided into the following groups: the miR-24 high expression, miR-24 interference (anti-miR-24), and blank control groups.
Proliferation of HUVECs was detected by the MTT assay (Solec Technology Co., Ltd., Beijing, China), according to the previously described method (9). Cells were planted onto the 96-well plate, at the density of 5 × 103 /well. When 50% confluence was reached, the cells were cultured with serum-free medium for 24 h. At 24 h, 48 h, and 72 h after transfection, 10 µL MTT was added into each well to incubate the cells for further 4 h. Then the medium was discarded, and 100 µL DMSO was added into each well. After shaking for 15 s, the absorbance at 570 nm (OD570 nm) was detected on a fluorescence detector. Experiments were performed in triplicates.
Scratch assay and Transwell chamber assay
Migration abilities of HUVECs were detected by the Scratch assay and Transwell chamber assay. For the Scratch assay (10), the cells were planted onto the 6-well plate, at the density of 4 × 105/well. After adherence, a short line was marked on the bottom of the plate with the pipette tip. Then the cells were washed with PBS (pH 7.4), and then cultured with serum-free 1640 medium. At 0 h and 24 h after this, the migration status was observed, and the short line width was measured. The migration ratio was calculated as follows: Migration ratio = (line width at 0 h - line width at 24 h) / line width at 0 h.
On the other hand, the migration ability of HUVECs was also detected by the Transwell chamber assay (3). The cells were planted on the upper chamber in the Transwell plate, at the density of 1 × 104/well, and cultured with serum-free 1640 medium. The lower chamber was added with 500 µL medium containing serum. After 12 h, the upper chamber was removed. The cells were carefully wiped off with a cotton swab, and fixed with 4% paraformaldehyde for 15 min. The upper chamber was washed with PBS, and stained with crystal violet at room temperature for 1 h. The cells in the lower chamber were observed with the inverted microscope. The five fields were randomly selected, and the cells were counted.
Tube formation capacity of HUVECs was examined by the Matrigel assay (Corning, Corning, NY, USA) (3). The 96-well plates were pre-coated with Matrigel (20 µL each well) on ice. Then the plated was placed in the 37 °C, 5% CO2 incubator for 1 h. The cells were seeded onto the Matrigel-coated plates, at the density of 1 × 104/well, and incubated for 12 h. Then five fields were randomly selected, and the small tubes were observed under microscope.
Total RNA was extracted with TRIzol. The first-strand DNA was synthesized with the following system: 4 µL 5 × reaction buffer, 1 µL RiboLock™ Ribonuclease Inhibitor (20 U/µL), 2 µL 10 mmol/L dNTP Mix, at 37 °C for 5 min, followed by adding 1 µL reverse transcriptase, at 42 °C for 1 h, and at 70 °C for 10 min. RT-PCR was performed with the PCR Kit from Tiangen Biotech, Beijing, China. The primer sequences were as follows: eNOS, forward 5’-AGGAACCTGTGTGACCCTCA-3’ and reverse 5’-CGAGGTGGTCCGGGTATCC-3’; Sp1, forward 5’-AGGTGCACCAGCTTCCAGGCCTG-3’ and reverse 5’-CCAGGTCCATGAAGGCCAAGTTG-3’; and β-actin, forward 5’-CTGGAACGGTGAAGGTGACA-3’ and reverse 5’-AAGGGACTTCCTGTAACAATGCA-3’. The PCR conditions were as follows: 94 °C for 5 min; 94 °C for 30 s, 56 °C for 1 min, 72 °C for 1 min, for totally 30 cycles; followed by 72 °C for 5 min. Totally 5 µL PCR products were subjected to the 1.5% agarose gel electrophoresis analysis.
Western blot analysis
Cell protein was lysed with lysis. The protein concentration was determined, and 30 µg protein was subjected to 8% SDS-PAGE, which was then electronically transferred onto the PVDF membrane. After blocking with non-fat milk for 1 h, the membrane was incubated with rabbit anti-human anti-eNOS primary polyclonal antibody (1:1000 dilution; Abcam, Cambridge, MA, UK) and rabbit anti-human anti-Sp1 primary polyclonal antibody (1:1000 dilution; Abcam), respectively, at 4 °C overnight. After washing with TBST (0.14 mol/L NaCl, 2.7 mmol/L KCl, 24.8 mmol/L Trise base, and 1% Tween; pH = 7.4). Then the membrane was incubated by the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2000 dilution; Santa Cruz, Santa Cruz, CA, USA), at room temperature for 1 h. The protein bands were scanned and analyzed by the Odyssey infrared fluorescence imaging system (Li-Cor Inc., Lincoln, NE, USA).
Data were expressed as mean ± SD. SPSS 16.0 software was used for statistical analysis. One-way ANOVA was performed for the group comparison, with the LSD-t and SNK-q tests. P < 0.05 was considered statistically significant.