1、MicroRNAs in Vascular Disease the beginning of a new taleChunxiang Zhang M.D.,Ph.D.University of Medicine and Dentistry of New Jersey.MiroRNAs MicroRNAs(miRNAs)are a novel class of endogenous,small,noncoding RNAs that regulate gene expression.Mature miRNAs are 18-to 24-nucleotide(nt)-long.They negat
2、ively regulate protein expression of their target mRNAs by either translation inhibition or degradation.As a group,miRNAs are estimated to regulate at least 30%of the human genes.Pathway of Genetic Information Flow and RNA“revolution”1.The standard pathway of information flow in a cell.2.DNA Transcr
3、iption and RNA products3.The first RNA revolution.Discovery of miRNA-The second RNA“revolution”Discovery of RNAi.With the help of RNAi technology,two regulatory noncoding RNAs were found,interfering RNAs(siRNA)and endogenous miRNAs.The recent discoveries of RNAi and miRNA represent the second RNA re
4、volution(Kong et al.Genomics Proteomics Bioinformatics.2005;3:62-72).miRNAs and siRNAs siRNAs and miRNAs have similar mechanism for gene expression regulation.However,they are different from each other.The chief difference lies in their origins:SiRNAs are produced from long double-stranded(bimolecul
5、ar)RNAs or long hairpins,often of exogenous origin.In contrast,miRNAs are endogenous.They are encoded within the genome and come from endogenous short hairpin precursor.Therefore,miRNAs are more important because they are endogenous regulators for gene expression.Biogenesis of miRNAs and their molec
6、ular mechanism for gene regulation.Biological functions of miRNAs Although only a small number from hundreds of identified miRNAs have been characterized,a growing body of exciting evidence suggests that miRNAs are important regulators for cell growth,differentiation,and apoptosis.Therefore,miRNAs c
7、ould be the important endogenous regulators in normal development,physiology,as well as in disease.miRNAs in diseases Consequently,dysregulation of miRNA function may lead to human diseases.In this respect,the most exciting research area is the role of miRNAs in cancer,because cell dedifferentiation
8、,growth,and apoptosis are important cellular events in the development of cancer.Indeed,both basic and clinical studies have demonstrated that miRNAs are aberrantly expressed in diverse cancers.The recent advances in the research of miRNAs and cancers have resulted in the following conclusions.First
9、,the aberrant expressions of miRNAs are tissue-and cancer-specific.Different tissues and cancers have different miRNA expression profiles.Second,multiple miRNAs are dysregulated in cancers.Third,miRNAs are thought to function as both tumor suppressors and oncogenes.Fourth,miRNAs may serve as novel t
10、herapeutic targets for cancer.Therefore,identifying the detailed functions of the key aberrantly expressed miRNAs is critical for cancer research .MiRNAs in cardiovascular disease Proliferative vascular disease has long been the leading cause of death in developed countries.Although miRNAs are highl
11、y expressed in the vascular system,the roles of these miRNAs in vascular disease are unknown.As vascular disease share some similar cellular events and molecular mechanisms with cancer,we hypothesize that miRNAs may play important roles in vascular disease.Animal Model Selection Vascular neointimal
12、lesion formation.Neointimal formation is the common pathological lesion in atherosclerosis,restenosis,diabetic vascular complication and transplant vascular disease.Hypothesis Cell growth(proliferation)and apoptosis are key cellular events in the formation of vascular neointimal lesion formation,whe
13、reas miRNAs are able to regulate these cellular events in other cell type.We therefore hypothesize that miRNAs may play important roles in vascular neointimal lesion formation by regulating vascular smooth muscle cell growth and/or apoptosis.MiRNAs in vascular neointimal lesion formation We are tryi
14、ng to answer the following questions to determine the potential role of miRNAs in vascular neointimal formation.Question#1:Are the miRNAs aberrantly expressed in vascular wall with neointimal lesion formation?Question#2:If the answer to the first question is yes,do these aberrantly expressed miRNAs
15、play a role neointimal lesion formation?Question#3:What are the cellular mechanisms responsible for miRNA-mediated effect on neoinimal formatiom?Question#4:What are the molecular mechanisms responsible for miRNA-mediated effect on neoinimal formatiom?.Rat carotid artery balloon injury To determine t
16、he expression changes of miRNAs in the vascular wall with neointimal formation,we applied the well-established rat carotid artery balloon injury model in my laboratory.In which,rat(Sprague-Dawley,250-300g)right common carotid artery injury was induced via a A 2F Fogarty balloon catheter.Remarkable n
17、eointimal lesion formation will be induced as early as 7 days after injury.MiRNA expression signature in normal rat carotid artery.MiRNAs are aberrantly expressed in rat carotid arteries after angioplasty Compared with normal uninjured arteries,microarray analysis demonstrated that aberrant miRNA ex
18、pression was a remarkable characteristic in vascular walls after angioplasty.Seven days after balloon injury,113 of the 140 artery miRNAs were differentially expressed with p-value 0.01;60 miRNAs were upregulated,and 53 miRNAs were downregulated.At 14 days after injury,110 of the 140 artery miRNAs w
19、ere differentially expressed(63 up and 47 down),while 102 of the 140 artery miRNAs were differentially expressed(55 up and 47 down)at 28 days after angioplasty.The time course changes of miRNAs that were highly expressed in rat carotid artery and over 1-fold upregulated after angioplasty.The time co
20、urse changes of miRNAs that were highly expressed in rat carotid artery and over 50%downregulated after angioplasty.Confirmation of the aberrant miRNA expression in injured arteries by qRT-PCR and/or Northern blot analysis.Do these aberrantly expressed miRNAs play a role in vascular neointimal lesio
21、n formation?To determine the potential role of these aberrantly expressed miRNAs in neointimal formation,we selected miR-21 as our first experimental target for the following reasons:miR-21 is one of the most up-regulated miRNAs after angioplasty.miR-21 is also up-regulated in diverse cancers.There
22、is a commercially available inhibitor.miR-21 inhibitor The antisense oligonucleotide for miR-21 is modified at each nucleotide by an O-methyl moiety at the 2-ribose position.The modified antisense oligonucleotide(2OMe-miR-21)is also called miRNA inhibitor.MiR-21 inhibitor(2OMe-miR-21)was synthesized
23、 by Integrated DNA Technologies and has the following sequence and structure:5mUmCmAmAmCmAmUmCmAmGmUmCmUmGmAmUmAmAmGmCmUmA-3.For the control purpose,we have used two controls for this study.The first control was a vehicle control(PBS)and the second control was an irrelevant 2-O-methyl-oligonucleotid
24、e(2OMe-EGFP).2OMe-EGFP was also synthesized by Integrated DNA Technologies and has the following sequence and structure:5mAmAmGmGmCmAmAmGmCmUmGmAmCmCmCmUmGmAmAmGmU-3.Local oligo delivery into the injured vascular wall.The effect of miR-21 on neointimal formation after angioplasty.What are the cellul
25、ar mechanisms involved in miR-21 mediated effect on neointimal formation?VSMC proliferation is the key cellular event for neointimal lesion formation.To identify the cellular mechanism involved in miR-21-mediated effect on neointimal lesion formation,we therefore determined the effect of miR-21 inhi
26、bitor 2OMe-miR-21 on cell proliferation in cultured VSMCs in the following experiments.It is well known that freshly isolated VSMCs mimic differentiated VSMCs in normal uninjured vascular wall,whereas serum cultured VSMCs mimic dedifferentiated VSMCs in vascular neointimal lesions.We thus determined
27、 the miR-21 levels in freshly isolated,differentiated VSMCs and dedifferentiated VSMCs cultured in DMEM containing 10%FBS.We found that the expression of miR-21 in dedifferentiated VSMCs was significantly higher than that in freshly isolated,differentiated VSMCs.2OMe-miR-21 decreases the miR-21 expr
28、ession level in cultured VSMCs in a dose-dependent manner.2OMe-miR-21 decreases VSMC proliferation in vitro.Representative BrdU stained photomicrographs(bottom panel)as well as their corresponding total cell photomicrographs(top panel).What is the effect of miR-21 on VSMC apoptosis?Neointimal growth
29、 is the balance between cell apoptosis and cell proliferation.Thus,apoptosis is also an important cellular event in neointimal lesion formation.Recent reports demonstrated that miR-21 had anti-apoptosis effect on glioblastoma cells,but had no anti-apoptosis effect on HeLa cells.Does miR-21 have anti
30、-apoptosis effect on VSMC apoptosis?To determine the effect of miR-21 on VSMC apoptosis,we applied a well-established VSMC apoptosis model in which apoptosis was measured after 48 h in serum-free culture.Apoptosis was evaluated by dUTP nick-end labeling(TUNEL)assay and caspase-3 activity measurement
31、.The effect of miR-21 on VSMC apoptosis in vitro.Representative TUNEL stained photomicrographs (bottom panel)as well as their corresponding total cell photomicrographs(top panel).2OMe-miR-21 decreases VSMC proliferation in vivo in balloon injured vessels.2OMe-miR-21 increases VSMC apoptosis in vivo
32、in balloon injured vessels.Potential gene targets in miR-21-mediated cellular effects on VSMCs miRNA is able to bind to its mRNA targets with either perfect or imperfect complementarity.Thus,one miRNA may have multiple mRNA targets.Based on the cellular effects of miR-21 on VSMCs,we proposed that mi
33、R-21 might target,degrade a tumor suppressor gene,and/or block its putational analysis of miR-21 targets Computational analysis suggests that PTEN,Bcl-2,and transforming growth factor-beta(TGF-beta)might be the potential target genes for miR-21.It is important that all the computational predicted mi
34、RNA targets should be verified by experimental approaches.TGF-beta is not a gene target for miR-21.Bcl-2 is not a direct target of miR-21.PTEN is a direct target of miR-21.Summary of the results1.miRNA expression signature in artery is identified for the first time.miRNAs are aberrantly expressed in
35、 the vascular walls with neointimal lesion formation.Modulating an aberrantly overexpressed miRNA,miR-21,via antisense-mediated depletion(knock-down)have a significant negative effect on neointimal lesion formation.In vitro,the expression level of miR-21 in dedifferentiated vascular smooth muscle ce
36、lls was significantly higher than that in fresh isolated,differentiated cells.Depletion of miR-21 resulted in decreased cell proliferation and increased cell apoptosis in a dose dependent manner.MiR-21-mediated cellular effects were further confirmed in vivo in balloon-injured rat carotid arteries.P
37、TEN and Bcl-2 are involved in miR-21-mediated cellulae effects,in which PTEN is a direct gene target of miR-21.Conclusion The results suggest that miRNAs are novel regulatory RNAs for neointimal lesion formation.MiRNAs may be a new therapeutic target for proliferative vascular diseases such as ather
38、osclerosis,postangioplasty restenosis,transplantation arteriopathy and stroke.Acknowledgments Postdoctoral fellows:Yunhui Chen M.D.,Ph.DRuirui Ji M.D.,Ph.DJian Yang M.D.Junming Yue Ph.DDavid B.Dean M.D.Xiaojun Liu Ph.D.He Chen Ph.D.Jawwad Yusuf M.D.Grants:R01 HL080133 R01 HL079004 R01 HL072902 R01 HL063886.Grant SupportNIH grants:R01 HL080133;R01 HL079004;R01 HL072902;R01 HL063886;R01 HL064165;R01 HL079109.AHA grant:AHA#0530106N.ADA grant:ADA#105JF60.Thank You!.
