| ObjectiveThe excess proliferation of vascular smooth muscle cells (VSMC) plays an important role in the pathology of cardiovascular hyperplasia diseases such as arthrosclerosis (AS), coronary heart disease and post-angioplasty restenosis (RS). In fact, superfluous proliferation is one of the major cellular events resulting in neointimal hyperplasia, vascular wall remodeling and RS. In present studies of the prevention and treatment of cardiovascular disease, regulation of proliferation, migration and apoptosis of VSMCs via gene therapy is a promising new avenue.Early growth response factor-1 (EGR-1) is a zinc finger transcription factor (TF), which controls the expression of multiple genes related to cell proliferation. EGR-1 expression is activated by an exogenous stimulus, facilitating cell proliferation and neointimal hyperplasia, which can then result in a pathological vascular repair reaction such as RS. Inhibiting the expression of EGR-1 may effectively block excessive VSMC proliferation, and ultimately prevent disease. The decoy oligodeoxynucleotides (ODNs) strategy is involves transfection of double-stranded ODNs, the sequence of which corresponds to the binding site sequence of the TF in question. These ODNs can compete for TF binding with the native TF binding site, thus leading to the inhibition of downstream gene regulation by the targeted TF. To investigate the mechanism of this inhibition and its effect on VSMC proliferation and apoptosis, we designed and synthesized decoy ODNs targeting EGR-1 and transfected them into the primary cultures of VSMCs in rats. We then evaluated ODN binding to EGR-1 and the subsequent effect on downstream gene expression, cell cycle, apoptosis and migration.Methods1. EGR-1 decoy oligodeoxynucleotides (decoy ODNs) and scrambled decoy ODNs (SCR) design and synthesisDouble-stranded EGR-1 decoy ODNs were synthesized by TaKaRa Biotech Co. (Dalian, China) according to the GenBank published, cis-element sequence of the EGR-1 binding site. The sequence is as follows:5'-TCGCCCTCGCCCCCGCTAAGGG-3',3'-AGCGGGGGCGGGGGCGATTCCC-5';The sequence of the scrambled ODNs (SCR) was:5'-AGCCGCACCGGCCTGCCTCGTC-3',3'-TCGGCGTGGCCGGACGGAGCAG-5'.3'and 5'phosphorothioate modifications were added to the ODNs to enhance their stability. The 5'terminus of a subset ODNs was labeled with fluorescein isothiocya-nate (FITC) to identify gene distribution under fluorescence microscopy.2. VSMC culture and experimental groupingAdherent culture was to be applied.Rat primary aortic smooth muscle cells were derived from the thoracic aorta medial explants of weighing 100-120g Wistar rats (Provided by China Medical University).The primary cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), pH7.4, containing 20% fetal bovine serum (FBS), 100U/ml penicillin and 100μg/ml streptomycin at 37℃in a humidified atmosphere of 5% CO2. Cells were passaged by washing once in phosphate buffered solution (PBS) followed by trypsinization. Subcultured strains were cultured in DMEM, pH7.4, containing 10% FBS, 100U/ml penicillin and 100μg/ml streptomycin. Cultured cells were identified as VSMCs by morphology and immunocytochemistry forα-SM-actin. Subcultured strains were used between passages 3 and 8.VSMCs were divided into three groups:the control group (normal culture cells), decoy group (transfection of EGR-1 decoy ODNs), SCR group (transfection of scrambled ODNs). Each group of cells were cultured in DMEM containing 10% FBS without antibiotics, the Decoy group was transfected with 0.1μmol/L Egr-1 decoy ODNs; the Decoy SCR group was transfected with 0.1μmol/L Egr-1 decoy ODNs SCR.3. ODNs transfectionVSMCs were cultured in DMEM containing 10% FBS without antibiotics, subconfluent VSMCs (70%) were growth-arrested in serum-free conditions for 30h before transfection with ODNs(0.1μmol/L) using FuGENE6. Cells were transfected a second time in the presence of 10% FBS 18h following the initial transfection. The transfected results were detected by fluorescence microscope and flow cytometry (FCM).Preparation of FuGENE6 Reagent:ODN complex:add corresponding FuGENE6 Transfection Reagent to the serum-free medium in 1.5ml sterile Eppendorf tubes, mix and incubate for 5 minutes at room temperature. Add ODNs to each tube respectively, using 3:1 ratio of FuGENE6 Transfection Reagent (μl) to ODN (μg), mix and incubate the transfection Reagent:ODNs complex for 15 minutes at room temperature, then add the complex to the cells.4. Electrophoretic Mobility Shift Assay (EMSA)Cells of every group were collected and the nucleoproteins were extracted according to the manufacturers instructions of Applygen Technologies Inc.(Beijing, China).4μg of nucleoprotein extraction were hybridized with 30 pmol biotin-labeled probe (TaKaRa Biotech Co. Dalian, China), according to the EMSA kit manufacturer's instructions (Roche).10μl of each reaction was electrophoresed on 6% native polyacrylamide gels (100 V,40 min) and transferred onto nitrocellulose filters (Amersham). Gels were exposed to develop in dark room by adding illumination (1:20 dilution, Viagene Biotech, Ningbo Co.). As a control, a mutation probe was added to prove the specific binding of decoy ODNs to EGR-1.5. Experiment methodsThe effect of ODN on proliferation of VSMC was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)metabolism measuring and 5-bromodeoxyuridine (BrdU) incorporation assay. FCM was performed to track cell cycle progression. Apoptosis in cultured VSMC was quantified by FCM and fluorescence microscope. The modified Boyden's chamber method (Transwell) and wound-healing assay were used to examine the abilities of VSMC migration. RT-PCR was done to detect the mRNA level of Egr-1, PCNA, TGF-β1, cyclinD1, CKD4, p53, p21, MMP-14, MMP-2 and TIMP-2. Western blot was performed to measure the protein expression of Egr-1, PCNA, TGF-β1, cyclinD1,CKD4, p53, p21, MMP-14, MMP-2 and TIMP-2.Results1. Tissue explants adherent method was used to culture VSMCs, a few of cells erupted 5 to 6 days later and approached confluence approximately 3 weeks. VSMCs were characterized by specific hill-and-valley appearance and by positiveα-SM-actin immunocytochemical staining. Highly purified primary VSMCs were gained and can be propagated about more than 20 passages. After 10 passages, the cells lost the proliferation ability gradually.2. The ODNs were readily detectable apparently within the nucleus and, to a lesser extent, the cytoplasm by the fluorescence microscope as a result of endocytosis. The green fluorescent could be seen localized in the nucleus of 71±1.25%,69±2.47%. VSMCs respectively on the 24th hour after transfectiOn of FITC labeled ODNs. Uptaking rate of FITC-Decoy ODNs and FITC-Decoy ODNs SCR detected by FCM was 13.9%,14.7% respectively.3. EMSA results revealed that transfected EGR-1 decoy ODNs can bind to EGR-1 protein and competitively occupy the DNA binding site. Accordingly, the amount of EGR-1 was markedly diminished in decoy ODN-treated nuclear extracts. In a word, the EGR-1-binding activity was effectively inhibited by EGR-1 decoy ODNs but not by SCR ODNs.4. The optical density (OD) of MTT decreased significantly in VSMCs transfected with Egr-1 decoy ODNs compared with that in VSMCs non-transfected. The inhibition ratio of VSMC proliferation was 22.8%,21.9%,20.7%respectively after transfecting of Egr-1 decoy ODNs on the 24th,48th and 72th hour. There was no significant change between the Decoy SCR and Control group. Egr-1 decoy ODNs can significantly inhibit 10% FBS induced VSMC proliferation.5. The labeling ratio of BrdU of the group control, Decoy and Decoy SCR was 39.30±4.51%,20.38±2.87%,38.47±3.55%respectively. Many proliferous cells were labeled by BrdU in the Control and Decoy SCR group, while the proliferous cells labeled by BrdU were obviously decreased in the Decoy group (P<0.01). There was no significant change between the Decoy SCR and Control group. The results of BrdU incorporation show that Egr-1 decoy ODNs can significantly inhibit the DNA synthesis in S phase, and inhibit 10% FBS induced VSMC proliferation.6. FCM analysis indicated that the G0/G1 phase fraction ratio of the Decoy group was higher than the Control group and Decoy SCR group, while its S-phase fraction ratio was lower than the Control group and Decoy SCR group, the proliferation index of the Decoy group decreased significantly. The result suggested that Egr-1 decoy ODNs blocked VSMC cycle in G0/G1 phase, inhibited the proliferation of VSMC.7. There was no significant difference about the ratio of apoptosis in VSMC among these groups by FCM analysis detected with Annexin V/FITC combined PI and fluorescence microscope detected with Hoechst33342/PI double labeled assay. 8. Wound-healing assay showed that the migration lenth of the group control, Decoy and Decoy SCR was 102.88±6.41,53.66±9.47,104.49±6.48μm respectively. The results suggested that EGR-1 decoy ODNs inhibited VSMC migration after mechanical injury (P<0.01). EGR-1 decoy ODNs SCR had no effect to VSMC migration.9. The modified Boyden's chamber method showed that the number of VSMC migration was reduced by 47.83%(P<0.01) at 24 hours after transfection of EGR-1 decoy ODNs. EGR-1 decoy ODNs SCR had no contribution to VSMC migration.10. Under TEM, the primary VSMCs of contractile phenotype were rich in cytoplasmic myofilaments, and little rough endoplasmic reticulum was observed. High passage VSMCs exhibited hypertrophy, increased mitochondia and rough endoplasmic reticulum, and a decrease in cytoplasmic myofilaments, indicating a synthetic phenotype. In the decoy ODN-transfected group, VSMCs had a contractile phenotype, with small nuclei, decreased organelles and a relatively normal amount of myofilaments. This indicates that EGR-1 decoy ODNs inhibited the transformation of VSMCs. SCR-transfected cells were similar to controls.11. The results of RT-PCR showed that the mRNA transcription of Egr-1, PCNA, cyclinDl, CDK4, MMP-14 and MMP-2 were decreased obviously in the Decoy group compared with the Control group and SCR group, while the mRNA transcription of p53, p21 and TIMP-2 had no significant change.12. The results of Western blot showed that protein translation of Egr-1, PCNA, cyclinD1, CDK4, MMP-14 and MMP-2 were decreased obviously in the Decoy group compared with the Control group and SCR group, while the protein translation of p53, p21 and TIMP-2 had no significant change.Conclusion1. Egr-1 Decoy ODNs gene transfection may specially suppress the expression of Egr-1 and corresponding genes with Egr-1. Egr-1 Decoy ODNs can significantly inhibit 10%FBS induced VSMC proliferation. This effect may be related to its blocking VSMC cell cycle in G0/G1 phase, inhibiting the DNA synthesis in S phase, decreasing the expression of cyclinDl, CDK4 and TGF-β1.2. Egr-1 Decoy ODNs can bind to EGR-1 protein and competitively occupy the DNA binding site. Accordingly, the amount of EGR-1 was markedly diminished in decoy ODN-treated nuclear extracts, then specially suppress the expression of Egr-1, regulate the expression of MMP-14, decrease activation of MMP-2, and inhibit VSMC migration.3. Egr-1 Decoy ODNs and Egr-1 Decoy ODNs SCR gene transfection does not influence the ratio of apoptosis in VSMC. |
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