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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 BackgroundoCal
2、cineurin/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-Dependen
3、t 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.Dalian 1.Calcineurin-Dependent Cardiac HypertrophyJeffery D. Molkentin et al. Cell,1998; 93,215228
4、. Aug.20-22,2009.Dalian 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
5、;101:2431-2437.Aug.20-22,2009.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 hypertrophyAu
6、g.20-22,2009.Dalian 5. Overexpression 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 cal
7、cineurin in cardiac hypertrophy? Calcineurin 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 Calcineur
8、in by miR-133. (A) The sequences showing the unique 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.
9、01 vs Control; # P0.05 vs miR-133. 00.51.0 Relative luciferase 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-UGAAC
10、AAGAUCCAAAAAACCAA-22463-UGUCGACCAACUUCCCCUGGUU-5Rat PP2B 3UTR (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 m
11、iR-133 level in cultured cardiomyocytes. *P0.01 vs non-transfection 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.
12、 Effects of miR-133 on calcineurin protein levels (n=5), mRNA level (n=5) and cell 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 l
13、evel (n=5) and cell area (The number of cells was in brackets) in cardiomyoctes treated with 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
14、in mice.Sham TAC TAC+CsA Aug.20-22,2009.Dalian A B Results: Calcineurin/NFAT Signaling Regulates 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
15、,2009.Dalian A B C Results: Calcineurin/NFAT Signaling Regulates miR-133 ExpressionFig.7. CsA restored the decreased 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/N
16、FAT Signaling Regulates miR-133 ExpressionFig.8. Primary rats cardiomyocytes in serum-free medium were stimulated with 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 Result
17、s: Calcineurin/NFAT Signaling Regulates miR-133 Expression Fig.9. CsA inhibited hypertrophy (The number was in brackets), calcineurin 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 S
18、ignaling Regulates miR-133 ExpressionFig.10. CsA alleviated the decreased miR-133 expression in PE-treated cardiomyocyes. *P0.01 vs Control. #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-1
19、33. (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
