The Effect Of Small Interfering RNA Against The AP-1 Gene On Proliferation, Migration, Differentiation, And Extracellular Matrix Turnover Of Vascular Smooth Muscle Cells

The effect of small interfering RNA(siRNA) against the AP-1 gent of proliferation,migration, differentiation,and extracellualr matrix turnover of rat arterial smooth muscle cellsPurposeRestenosis is the most important barrier to the long-term success of percutaneous transluminal coronary angioplasty。While the mechanisms of restenosis are poorly understood,many processes contribute,including thrombus formation,vascular smooth muscle cell(VSMC) migration and proliferation,acute recoil,and chronic remodeling with extracellular matrix accumulation.Each component is a potential target for therapy. Postmortem studies reveal that restenosis is due at least in part to neointimal formation.The proliferation and migration of vascular smooth muscle cells(VSMC) may play a key role in the development of intimal thickening after arterial wall injury or in atherosclerosis。VSMC in the media has low mitogenic activity.During the early stages of arterial wall injury or atherosclerosis,aortic smooth muscle cells may undergo transition from a contractile to a synthetic phenotype and begin proliferating in response to various growth factors,causing intimal thickening of the arterial walls.Phenotypic modulation of vascular smooth muscle cells(SMCs) from contractile to synthetic forms plays a pivotal role in the pathogenesis of vascular diseases including atherosclerosis. However,in addition to growth factor stimulation,the replication and migration of VSMC may require the degradation or remodeling of extracellular matrix surrounding the cells。VSMC synthesizes important components of the extracellular matrix,including collagens,elastin,and proteoglycans. An imbalance between the accumulation and degradation of extracellular matrix may be crucial in the development of intimal thickening that forms after vascular wall interventions Matrix remodeling requires the action of proteinases,among which the matrix metalloproteinases(MMPs) appear to play a key role。AP-1 DNA binding activity is significantly increased in balloon-injured arteries.Much of our current knowledge about the characteristics of transcription factors comes from the discovery and study of activating protein 1(AP-1).AP-1 collectively describes a group of structurally and functionally related members of the Jun protein family[Jun(originally described as c-Jun),JunB and JunD]and Fos protein family[Fos (originally described as c-Fos),FosB,Fra-1 and Fra-2].Additionally,some members of the ATF(ATFa,ATF-2 and ATF-3) and JDP(JDP-1 and JDP-2) subfamilies,which share structural similarities and form heterodimeric complexes with AP-1 proteins (predominantly with Jun proteins),can bind to TRE-like sequences.Each of these proteins is differentially expressed and regulated,which means that every cell type has a complex mixture of AP-1 dimers with subtly different functions.Massive researches demonstrated that the existence of the affinitive site of AP-1 with the DNA,which is possibly one of the important mechanisms of some specific gene inducing AP-1 combination.Therefore,we reduced the total DNA combining activity of AP-1 through either AP-1 ODN or antisense oligonucleotide of c-Jun or c-Fos to suppress smooth muscle cell proliferation,all of which obtained the obvious effect.At present,antigene therapy focusing on the inhibition of VSMC proliferation has emerged as a potentially attractive strategy for reducing restenosis after PTCA. However,in vivo usage of decoy ODN is hampered by nuclease digestion.Stealth^TM RNAi overcomes the problem and remains stable for up to 72 hours making it a better choice for projects that involve work with animal models.RNA interference was a new method of fast closure gene which developed recently.when cell was introduced with homology double strands RNA(dsRNA) with endogenous mRNA coding region,this mRNA proceed the degeneration to cause the gene silence.The phenomenon is called the post-transcription gene silence.The small fragment interference RNA composed with 21-25 nucleotides is the molecular which has an effect directly in the RNAi process,and the cell displays the depletion of the specific phenotype.However,RNAi does not have any influence to the chromosome DNA sequence duplication and the transcription process.We supposed that gene silence can cause the AP-1 compound activity to weaken or to lose.We designed rat AP-1 siRNA,and respectively set up target sites in the two subunits which were c-Jun and c-Fos using Lipofectamine^TM20OO as transfection reagent.We transfected AP-1 siRNA into vascular smooth muscle cell to detect whether the proliferation,migration and dedifferentiation changed by suppressing the gene expression of AP-1 and whether VSMCs with AP-1 gene silence changed correspondingly with serum stimulation.Methods1,We did the primary cell culture through the tissue adherence method,Under the inverted microscope,we observed that the growth of the cells took on the the the smooth muscle cell characteristic shapes of "the peak" and "the valley" Mouse anti-rat monoclonal antibody aboutα-SM-actin was used for the examination of immunofluorescence.2,siRNAs Three pairs of Stealth^TM siRNA were designed and synthesized by the Invitrogen company in America.At the meantime,AP-1 gene independent siRNA was synthesized as the negative control siRNA.We input the specific siRNA sequences to NCBI database to blast,and the results showed that EST sequence didn't have the homology with other genes.Prepared the Stealth^TM RNA transfection solution,and added the corresponding tranfection solution in every group(no transfection in normal control group,the transfection of negative siRNA in negative siRNA group,the transfections of AP-1 +A,AP-1 +B and AP-1 + C siRNAs respectively in positive siRNA groups).3,Using RT-PCR analysis,effects of AP-1 siRNA on mRNA expression of AP-1 were examined while the phosphorylation levels of c-Jun and c-Fos and activity of Ap-1 were determined by Western blotting and AP-1 DNA-binding activity assay4,Effects of AP-1 siRNA on rat VSMC cells in vitro were determined using the cell cycle analysis,MTT-tests,and PCNA expression.simultaneously examined the migration ability of smooth muscle cell with a modified Boyden-chamber assay5,we observated the expression of SMα-actin by RT-PCR analysis and Western blotting,we observed differentiation of smooth muscle cell by Immunofluorescence.6,Using RT-PCR analysis,effects of AP-1siRNA on mRNA expression of MMP-2 were examined while t MMP-2 and aTIMP-2 were determined by Western blotting.MMP-2 and MMP-9activity in conditioned medium of cultured VSMCs was analyzed by substrate-gel electrophoresis(zymography).Results1,After preliminary transfections with AP-1 siRNA containing the 3 different target sequences,we chose the one group for AP-1 +B siRNA because it produced the maximal inhibition in protein level and mRNA.VCMCs were transfected with AP-1-targeted siRNA and nonsilencing control siRNA.with a very high level of AP-1 DNA binding activity in the mock cells,decreased AP-1 DNA binding activity was evident in the cells treated with AP-1+BsiRNA.2,cell DNA content was measured by FACS to determine the cell cycle distribution under various culture conditions.Cells were initially deprived of FBS for 24 h and subsequently incubated with serum for 24 h.The treatment of serum stimulated VSMC with AP-1+BsiRNA for 48 h,followed by staining with propidium iodide and subsequent flow cytometric analysis,demonstrated that compared to the non-transfected(mock) or control siRNA VSMCs,AP-1siRNA induced a significant accumulation of cells in the G1 phase of the cell cycle,suggesting that the observed growth inhibitory effects of AP-1siRNA in VSMC were due to cell cycle arrest.The antiproliferative effect of AP-1siRNA in rat VSMC was assessed by cell counting using the MTT assay.Proliferation of cells treated with AP-1siRNA(100nm) was attenuated.after transfaction,VSMC was incubated for 24 h with AP-1siRNA and AP-1siRNA caused a significant inhibition of VSMC proliferation.control siRNA had no significant effect on cell proliferation.The pattern of distribution of PCNA-positive cells among VSMC was observed for the total cell number.The highest number of proliferating cells was found(2-fold higher as compared with AP-1+BsiRNA group VSMC).Significant decrease of PCNA positive cells was revealed in AP-1+BsiRNA group by comparison with mock and control RNA VSMC,AP-lsiRNA inhibited smooth muscle cell proliferation,(P＜0.05).control siRNA had no significant effect on cell proliferation3,The expression of c-myc,bFGF,ET-1,TGF-β1mRNA was 0.63,0.68.0.61and 0.70 times smaller,respectively,in AP-1 +B siRNA transfection group than in the non-transfected(mock)group.On the other hand,the expression levels of c-myc,bFGF, ET-1,TGF-β1 showed significant decrease already after AP-1 +B siRNA transfection,and did not change in control siRNA VSMCs.4,To elucidate the role of AP-1siRNA on the differentiation of SMCs,the expressional pattern of differentiation-related gene in response to AP-1siRNA were monitored with the VSMC cells transfected with AP-1+BsiRNA.The levels and mRNA of SMα-actin were elevated and increased in the AP-1+BsiRNA-transfected VSMC cells compared to the non-transfected(mock).Control siRNA had no significant effect on the expression of SMα-actin.Additionally,the shape of AP-1+BsiRNA-transfected VSMC cells was turned into a spindle-like form,a characteristic phenotype of differentiated SMCs,indicating that the AP-1 siRNA might induce the cellular alteration.5,Boyden chamber migration assays were performed on rat aortic smooth muscle cells,and cells were counted under light microscopy.AP-1siRNA inhibited smooth muscle cell migration and reduced smooth muscle cell migration to 41 of the non-transfected(mock),(P＜0.05).control siRNA had no significant effect on cell migration.6,evidences the effect of AP-1siRNA in cell supernatants from cultured VSMC.A single band corresponding to a MMP-2 active form was evidenced in mock group both by zymography and Western blot;AP-1siRNA decreased the density of MMP-2 band as far as Western blot is concerned).In contrast to mock group,the basal secretion of pro-MMP-2 and the serum-induced activation of MMP-2 were not affected by control siRNA.1,These results indicate that anti-AP-1 siRNA effectively and specifically down-regulated AP-1 mRNA and c-Jun and c-Fos protein expression in VCMCs.,.suppressed the expression of the downstream genes of AP-l,and decreased AP-1 DNA binding activity.2,Our data demonstrate that AP-1 siRNA prevents VSMC proliferation and migration and promotes differentiation in vitro.3,The activity of MMP-2 was inhibited by AP-1 siRNA,MMP-2/TIMP-2 ratios incline.,decreased the accumulation of extracellular matrix.