Nrdp1 siRNA重组腺病毒载体的构建及其对心肌肥大的影响
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摘要
目的构建及鉴定神经调节素受体蛋白1(Nrdp1)基因siRNA重组腺病毒载体,初步研究Nrdp1基因对心肌细胞肥大的影响。方法设计并合成Nrdp1的siRNA靶向DNA序列,克隆至穿梭载体GV119中,并与腺病毒骨架质粒Ad Max在BJ5183细菌中进行同源重组,转染HEK293细胞,包装得到含si Nrdp1的重组腺病毒,实时定量PCR及Western blot检测重组腺病毒对新生大鼠原代心肌细胞Nrdp1表达的影响。然后利用血管紧张素Ⅱ和沉默Nrdp1的腺病毒干预体外培养的新生大鼠原代心肌细胞,实时定量PCR检测心肌肥大标志基因(ANF、β-MHC、Skeletal-α-actin)的表达。结果酶切后PCR分析、测序鉴定表明,干扰Nrdp1腺病毒构建成功,病毒纯化后滴度为1.5E+9 PFU/m L;重组干扰Nrdp1腺病毒可在蛋白水平和mRNA水平明显抑制Nrdp1的表达(P<0.001);沉默Nrdp1基因后,可明显上调血管紧张素Ⅱ诱导的心肌细胞肥大标志基因ANF、β-MHC和Skeletal-α-actin的表达(P<0.01)。结论构建的Nrdp1 siRNA重组腺病毒能有效地抑制新生大鼠原代心肌细胞中Nrdp1表达,且Nrdp1基因沉默后可加重AngⅡ诱导的心肌细胞肥大。
Aim To construct and identificate recombinant adenovirus with si Nrdp1 gene using Ad Max system,and investigate the effect of Nrdp1 gene on cardiomyocyte hypertrophy. Methods Designing and synthesizing siRNA sequences targeting of Nrdp1 DNA,then cloned into the shuttle vector GV119 and homologous recombinated with adenovirus backbone plasmid Ad Max in BJ5183 bacteria transfected HEK293 cells,and got adenovirus containing Nrdp1-siRNA gene through packaging. Real-time quantitative PCR and Western blot were used to detect Nrdp1 expression in primary rat neonatal cardiomyocytes. After adenoviral containing si Nrdp1 transfection and angiotensin Ⅱ( AngⅡ) stimulation,realtime quantitative PCR was used to detect the expression of myocardial hypertrophy marker gene( ANF,β-MHC and Skeletal-α-actin) of rat neonatal cardiomyocytes. Results Digested PCR analysis and sequencing showed that interference Nrdp1 adenovirus was successfully constructed,and the titer of virus was 1. 5E + 9 PFU / m L. Real-time PCR and Western blot indicated that the expressions of Nrdp1 mRNA and protein were greatly inhibited after infection in rat primary cardiomyocytes with recombinant adenovirus particles( P < 0. 001). Nrdp1 gene silencing cloud significantly increase expression of AngⅡ induced cardiomyocyte hypertrophy marker genes including ANF,β-MHC and Skeletal-α-actin( P <0. 01). Conclusion The recombinant adenovirus vector containing the Nrdp1-siRNA gene was successfully constructed,which can effectively silence Nrdp1 gene and enhance AngⅡ induced cardiomyocytes hypertrophy in vitro.
Aim To construct and identificate recombinant adenovirus with si Nrdp1 gene using Ad Max system,and investigate the effect of Nrdp1 gene on cardiomyocyte hypertrophy. Methods Designing and synthesizing siRNA sequences targeting of Nrdp1 DNA,then cloned into the shuttle vector GV119 and homologous recombinated with adenovirus backbone plasmid Ad Max in BJ5183 bacteria transfected HEK293 cells,and got adenovirus containing Nrdp1-siRNA gene through packaging. Real-time quantitative PCR and Western blot were used to detect Nrdp1 expression in primary rat neonatal cardiomyocytes. After adenoviral containing si Nrdp1 transfection and angiotensin Ⅱ( AngⅡ) stimulation,realtime quantitative PCR was used to detect the expression of myocardial hypertrophy marker gene( ANF,β-MHC and Skeletal-α-actin) of rat neonatal cardiomyocytes. Results Digested PCR analysis and sequencing showed that interference Nrdp1 adenovirus was successfully constructed,and the titer of virus was 1. 5E + 9 PFU / m L. Real-time PCR and Western blot indicated that the expressions of Nrdp1 mRNA and protein were greatly inhibited after infection in rat primary cardiomyocytes with recombinant adenovirus particles( P < 0. 001). Nrdp1 gene silencing cloud significantly increase expression of AngⅡ induced cardiomyocyte hypertrophy marker genes including ANF,β-MHC and Skeletal-α-actin( P <0. 01). Conclusion The recombinant adenovirus vector containing the Nrdp1-siRNA gene was successfully constructed,which can effectively silence Nrdp1 gene and enhance AngⅡ induced cardiomyocytes hypertrophy in vitro.
引文
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[7]许研,刘海梅,徐进文,等.ERK1/2蛋白在17β-雌二醇抑制睾酮诱导的心肌细胞肥大反应中的作用[J].中国动脉硬化杂志,2012,20(10):876-880.
[8]Zeng Y,Wang HX,Guo SB,et al.Transcriptional effects of E3 ligase atrogin-1/MAFbx on apoptosis,hypertrophy and inflammation in neonatal rat cardiomyocytes[J].PLo S One,2013,8(1):e53831.
[9]Maejima Y,Usui S,Zhai P,et al.Muscle-specific RING finger 1negatively regulates pathological cardiac hypertrophy through downregulation of calcineurin A[J].Circ Heart Fail,2014,7(3):479-490.
[10]Schisler JC,Rubel CE,Zhang C,et al.CHIP protects against cardiac pressure overload through regulation of AMPK[J].J Clin Invest,2013,123(8):3 588-599.
[11]Zhang Y,Zeng Y,Wang M,et al.Cardiac-specific overexpression of E3 ligase Nrdp1 increases ischemia and reperfusion-induced cardiac injury[J].Basic Res Cardiol,2011,106(3):371-383.
[12]Zhang Y,Tian C,Du J,et al.Overexpression of Nrdp1 in the heart exacerbates doxorubicin-induced cardiac dysfunction in mice[J].PLo S One,2011,6(6):e21104.
[2]Abdullah JM,Li X,Nachtman RG,et al.FLRF,a novel evolutionarily conserved RING finger gene,is differentially expressed in mouse fetal and adult hematopoietic stem cells and progenitors[J].Blood Cells Mol Dis,2001,27(1):320-333.
[3]Ingalla EQ,Miller JK,Wald JH,et al.Post-transcriptional mechanisms contribute to the suppression of the Erb B3 negative regulator protein Nrdp1 in mammary tumors[J].J Biol Chem,2010,285(37):28 691-697.
[4]Qiu XB,Markant SL,Yuan J,et al.Nrdp1-mediated degradation of the gigantic IAP,BRUCE,is a novel pathway for triggering apoptosis[J].EMBO J,2004,23(4):800-810.
[5]Wang C,Chen TY,Zhang J,et al.The E3 ubiquitin ligase Nrdp1‘preferentially’promotes TLR-mediated production of type I interferon[J].Nat Immunol,2009,10(7):744-752.
[6]陈志衡,杨作成.PI3K-Akt-m TOR信号通路与病毒性心肌炎[J].国际病理科学与临床杂志,2012,32(3):226-230.
[7]许研,刘海梅,徐进文,等.ERK1/2蛋白在17β-雌二醇抑制睾酮诱导的心肌细胞肥大反应中的作用[J].中国动脉硬化杂志,2012,20(10):876-880.
[8]Zeng Y,Wang HX,Guo SB,et al.Transcriptional effects of E3 ligase atrogin-1/MAFbx on apoptosis,hypertrophy and inflammation in neonatal rat cardiomyocytes[J].PLo S One,2013,8(1):e53831.
[9]Maejima Y,Usui S,Zhai P,et al.Muscle-specific RING finger 1negatively regulates pathological cardiac hypertrophy through downregulation of calcineurin A[J].Circ Heart Fail,2014,7(3):479-490.
[10]Schisler JC,Rubel CE,Zhang C,et al.CHIP protects against cardiac pressure overload through regulation of AMPK[J].J Clin Invest,2013,123(8):3 588-599.
[11]Zhang Y,Zeng Y,Wang M,et al.Cardiac-specific overexpression of E3 ligase Nrdp1 increases ischemia and reperfusion-induced cardiac injury[J].Basic Res Cardiol,2011,106(3):371-383.
[12]Zhang Y,Tian C,Du J,et al.Overexpression of Nrdp1 in the heart exacerbates doxorubicin-induced cardiac dysfunction in mice[J].PLo S One,2011,6(6):e21104.