1、Reciprocal Repression between MicroRNA-133 and Calcineurin Controls Cardiac Hypertrophy:A Novel Mechanism for Progressive Cardiac HypertrophyDe-Li Dong,Yan-Jie Lu,Bao-Feng Yang(董德利董德利 吕延杰吕延杰 杨宝峰杨宝峰)Department of PharmacologyHarbin Medical University,P.R.China Aug.20-22,2009.Dalian BackgroundoCalcine
2、urin/NFAT pathway induces Cardiac Hypertrophy oCalcineurin activation in cardiac hypertrophyoDecreased miR-133 expression in cardiac hypertrophy oOverexpression of miR-133 inhibits cardiac hypertrophy oInhibition of miR-133 induces cardiac hypertrophyAug.20-22,2009.Dalian 1.Calcineurin-Dependent Car
3、diac HypertrophyJeffery D.Molkentin et al.Cell,1998;93,215228.Aug.20-22,2009.Dalian 1.Calcineurin-Dependent Cardiac HypertrophyJeffery D.Molkentin et al.Cell,1998;93,215228.Aug.20-22,2009.Dalian 1.Calcineurin-Dependent Cardiac HypertrophyJeffery D.Molkentin et al.Cell,1998;93,215228.Aug.20-22,2009.D
4、alian Overexpression of calcineurin in mouse causes sudden cardiac death associated with decreased density of K channelsDeli Dong.et al.Cardiovascular Research 57(2003)320332Aug.20-22,2009.Dalian 2.Calcineurin Activation in cardiac hypertrophyHae W.Lim et al.Circulation.2000;101:2431-2437.Aug.20-22,
5、2009.Dalian 3.Calcineurin/NFAT Coupling Participates in Pathological,butnot Physiological,Cardiac HypertrophyWilkins BJ,et al.Circ Res.2004;94:110-118.Aug.20-22,2009.Dalian Car A et al.Nat Med.2007;13(5):613-8.4.Decreased miR-133 expression in cardiac hypertrophyAug.20-22,2009.Dalian 5.Overexpressio
6、n of miR-133 inhibits cardiac hypertrophyCar A et al.Nat Med.2007;13(5):613-8.Aug.20-22,2009.Dalian 6.Inhibition of miR-133 induces cardiac hypertrophyCar A et al.Nat Med.2007;13(5):613-8.Aug.20-22,2009.Dalian Hypothesis:Interaction between miR-133 and calcineurin in cardiac hypertrophy?Calcineurin
7、is the target of miR-133;The expression of miR-133 is regulated by calcineurin/NFAT signaling pathway.CalcineurinmiR-133Cardiac hypertrophyAug.20-22,2009.Dalian Results 1.miR-133 Targets Calcineurin Figure 1.Post-transcriptional Repression of Calcineurin by miR-133.(A)The sequences showing the uniqu
8、e sites of miRNA:mRNA complementary between miR-133 and calcineurin for rat and mouse genes.(B)Co-transfection of miR-133 with the luciferase reporter gene linked to the 3UTR of calcineurin reduced luciferase activity in rat and mouse species.*P0.01 vs Control;#P0.05 vs miR-133.00.51.0 Relative luci
9、ferase activity 00.51.0NCmiR-133+AMO-133miR-133AMO-133Mut-133*#*BMouse(n=8)Rat(n=10)5-UUGGUCCCCUUCAACCAGCUGU-3mmu-miR-133a/rno-miR-133aRat PP2B 3UTR(NM_017042)Rat miR-1333-UGUCGACCAACUUCCCCUGGUU-51600-AACCAUAGUGCCCAGUGACCAG-1621 2225-UGAACAAGAUCCAAAAAACCAA-22463-UGUCGACCAACUUCCCCUGGUU-5Rat PP2B 3UTR
10、(NM_017042)Site 1Site 2Mouse PP2B 3UTR(NM_008914)Mouse miR-1333-UGUCGACCAACUUCCCCUGGUU-51739-AGGAGCUGGAGGGGUUGACCAA-1760AAug.20-22,2009.Dalian Results:miR-133 Targets CalcineurinFig.2.(A)transfection of miR-133 specifically increased miR-133 level in cultured cardiomyocytes.*P0.01 vs non-transfectio
11、n group.(B)The negative control(NC)had no effect on miR-133 level,but AMO-133 decreased miR-133 level.*P0.01 vs control(lipofectamine transfection agents).n=3.ABAug.20-22,2009.Dalian Results:miR-133 Targets Calcineurin Fig.3.Effects of miR-133 on calcineurin protein levels(n=5),mRNA level(n=5)and ce
12、ll area(The number was in brackets)in normal cultured cardiomyoctes.*P0.01 vs Control.Aug.20-22,2009.Dalian A B C 1.miR-133 Targets CalcineurinFigure 4.Effects of miR-133 on calcineurin protein levels(n=5),mRNA level(n=5)and cell area(The number of cells was in brackets)in cardiomyoctes treated with
13、 PE(50M).A B C Aug.20-22,2009.Dalian Results:Calcineurin/NFAT Signaling Regulates miR-133 ExpressionFig.5.Calcineurin inhibitor CsA inhibited cardiac hypertrophy induced by transverse aortic constriction in mice.Sham TAC TAC+CsA Aug.20-22,2009.Dalian A B Results:Calcineurin/NFAT Signaling Regulates
14、miR-133 ExpressionFig.6.Calcineurin inhibitor CsA inhibited cardiac hypertrophy(The number was in brackets),calcineurin expression(n=5)and activity(n=5).*P0.01 vs Control;#P0.01 vs TAC.Aug.20-22,2009.Dalian A B C Results:Calcineurin/NFAT Signaling Regulates miR-133 ExpressionFig.7.CsA restored the d
15、ecreased miR-133 expression but not the increased miR-195 expression in TAC-induced hypertrophic heart.*P0.01 vs Control.#P0.01 vs TAC;n=5.Aug.20-22,2009.Dalian A B Results:Calcineurin/NFAT Signaling Regulates miR-133 ExpressionFig.8.Primary rats cardiomyocytes in serum-free medium were stimulated w
16、ith PE and PE+CsA.Cardiomyocytes were identified with a-actinin antibody(red signal)and nuclei were stained with bis-benzamide(blue).ControlPEPE+CsAAug.20-22,2009.Dalian A B Results:Calcineurin/NFAT Signaling Regulates miR-133 Expression Fig.9.CsA inhibited hypertrophy(The number was in brackets),ca
17、lcineurin expression(n=5)and activity(n=5)in cardiomyocytes treated with PE(50M).*P0.01 vs Control.#P0.01 vs PE.Aug.20-22,2009.Dalian A B C Results:Calcineurin/NFAT Signaling Regulates miR-133 ExpressionFig.10.CsA alleviated the decreased miR-133 expression in PE-treated cardiomyocyes.*P0.01 vs Cont
18、rol.#P0.05 vs PE;n=5.Aug.20-22,2009.Dalian Results:Calcineurin/NFAT Signaling Regulates miR-133 ExpressionFig.11.Calcineurin/NFAT-dependent Regulation of miR-133.(A)Antisense-A ODNs increased the expression of miR-133 in cultured cardiomyocytes.*P0.01 vs Control;n=4.(B)NFAT decoy ODNs increased the expression of miR-133 in cultured cardiomyocytes.*P0.01 vs Control;n=5.Aug.20-22,2009.Dalian Conclusion:Aug.20-22,2009.Dalian Chang Chen,Rong Huo,Ning Wang,Zhe Li,Yu-Jie Tu,AcknowledgmentsJun-Tao Hu,Xia Chu,Wei Huang,Yan-Jie LuBao-Feng YangAug.20-22,2009.Dalian
