DNAenzyme Targeting Egr-1 Inhibits Rat Vascular Smooth Muscle Cell Proliferation And Migration By Downregulation Of Cyclin D1, TGF-β1 And MMPs

ObjectivePercutaneous coronary intervention(PCI) is now one of widely used methods to treat coronary artery disease,but restenosis following PCI during 6 months limits its long-term benefits.The biological mechanisms are complex,involving elastic recoil in the first instance,followed by extracellular maxtrix production,smooth muscle cell proliferation and migration,and remodeling of the vessel.Therefore the hunt for efficient inhibitors of restenosis remains an ongoing challenge.Deoxyribozyme(DRz) is a DNA molecule with the activity of enzyme catalysis(DNAenzyme),which has practical therapeutic implications as a new category of gene-inhibitor.Early growth response factor-1(Egr-1)is a zinc finger structural factor that is activated under the outside stimulation of arterial injury leading to the splitting,migration and proliferation of vascular smooth muscle cell(VSMC)and neointimal hyperplasia.The aim of this study was to generate a novel DNAenzyme targeting Egr-1(10-23DRz,ED5)and a scrambled ED5(ED5SCR)as control to observe the effect of ED5 on proliferation, apoptosis and migration of rat vascular smooth muscle cells(VSMCs)and study its mechanism of inhibiting proliferation and migration.Methods1.Oligodeoxynucleotide(ODN) synthesisED5 and ED5SCR were synthesized by Takara and the base sequence of ED5 was:5'-CCGCTGCCAGGCTAGCTACAACGACCCGGACGT-3',ED5SCR:5'-GCCA GCCGCGGCTAGCTACAACGATGGCTCCAC-3',recognition sequence underlined; catalytic domain in the middle.ODN was modified by phosphorothioic acid at the 3' end and 5' end for increasing stability,and a small part was labelled with fluorescein isothiocyanate(FITC) at the 5' end for visualization of cellular uptake of ODN after transfection under fluorescence microscope.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 120-150g 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 idenfied 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,the ED5-treated group and the ED5SCR-treated group according to ODN used.Each group of cells were cultured in DMEM containing 10%FBS without antibiotics,the ED5 group was transfected with 0.1μmol/L ED5;the ED5SCR group was transfected with 0.1μmol/L ED5SCR.3.ODN transfectionVSMCs were cultured in DMEM containing 10%FBS without antibiotics, subconfluent VSMCs(70%) were growth-arrested in serum-free conditions for 30h before transfecting with ODN(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 ODN to each tube respectively, using 3:1 ratio of FuGENE6 Transfection Reagent(μl) to ODN(μg),mix and incubate the transfection Reagent:ODN complex for 15 minutes at room temperature,then add the complex to the cells.4.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,p53, p21,Bax,MMP-14,MMP-2 and TIMP-2.Western blot was performed to measure the protein expression of Egr-1,PCNA,TGF-β1,p53,p21,Bax,MMP-14,MMP-2 and TIMP-2.The protein expression of Egr-1,cyclin D1 was measured by immunocytochemistry.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 24 population doublings(PD).After PD 10,the cells lost the proliferation ability gradually.2.The ODN was readily detectable apparently within the cytoplasm and,to a lesser extent,the nucleus by the fluorescence microscope as a result of endocytosis. The green fluorescent could be seen localized in the cytoplasm of 70.6±1.52%,72±2.73%VSMCs respectively on the 24th hour after transfecting of FITC labelled ED5 and ED5SCR.Uptaking rate of FITC-ED5 and FITC-ED5SCR detected by FCM was 13.5%,15.2%respectively.3.The optical density(OD) of MTT decreased significantly in VSMCs transfected with ED5 compared with that in VSMCs non-transfected.The inhibition ratio of VSMC proliferation was 25%,20%,18%respectively after transfecting of ED5 on the 24th,48th and 72th hour.There was no significant change between the ED5SCR and control group.ED5 can significantly inhibite 10%FBS induced VSMC proliferation. 4.The labeling ratio of BrdU of the group control,ED5 and ED5SCR was 30.46±4.38%,15.37±2.32%,29.17±4.15%respectively.Many proliferous cells were labelled by BrdU in the control and ED5SCR group,while the proliferous cells lablled by BrdU were obviously decreased in the ED5 group(P＜0.01).There was no significant change between the ED5SCR and control group.The results of BrdU incorporation show that ED5 can significantly inhibite the DNA synthesis in S phase, and inhibite 10%FBS induced VSMC proliferation.5.FCM analysis indicated that the G₀/G₁ phase fraction ratio of the ED5 group was higher than the control group and ED5SCR group,while its S-phase fraction ratio was lower than the control group and ED5SCR group,the proliferation index of the ED5 group decreased significantly.The result suggested that ED5 blocked VSMC cycle in G₀/G₁ phase,inhibited the proliferation of VSMC.6.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 labelled assay.7.Wound-healing assay showed that the migration lenth of the group control,ED5 and ED5SCR was 159.62±9.57,65.35±5.04,162.84±8.43μm respectively.The results suggested that ED5 inhibited VSMC migration after mechanical injury(P＜0.01). ED5SCR had no effect to VSMC migration.8.The modified Boyden's chamber method showed that the number of VSMC migration was reduced by 32.18%at 6 hour after transfecting of ED5.ED5SCR had no contribution to VSMC migration.9.The results of RT-PCR showed that the mRNA transcription of Egr-1,PCNA, TGF-β1,MMP-14 and MMP-2 were decreased obviously in the ED5 group compared with the control group and ED5SCR group,while the mRNA transcription of p53,p21, Bax and TIMP-2 had no significant change.10.The results of Western blot showed that protein translation of Egr-1,PCNA, TGF-β1,MMP-14 and MMP-2 were decreased obviously in the ED5 group compared with the control group and ED5SCR group,while the protein translation of p53,p21 and TIMP-2 had no significant change.The result of Bax was not shown.11.The result of immunocytochemistry showed that protein translation of Egr-1, cyclin D1 was decreased obviously in the ED5 group compared with the control and ED5SCR group.ED5SCR had no effect to the protein translation of Egr-1,cyclin D1.Conclusion1.ED5 gene transfection may specially suppress the expression of Egr-1 and corresponding genes with Egr-1.ED5 can significantly inhibite 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 cyclin D1 and TGF-β1.ED5SCR gene transfection does not have the same effect as ED5.2.ED5 gene transfection may specially suppress the expression of Egr-1,regulate the expression of MMP-14,decrease activation of MMP-2,and inhibit VSMC migration.ED5SCR gene transfection does not have the same effect as ED5.3.ED5 and ED5SCR gene transfection does not influence the ratio of apoptosis in VSMC.