ALK-3、Pax-8基因抑制后大鼠心肌细胞中Smad1/5/8的表达
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摘要
目的:抑制大鼠胚胎心肌细胞株中ALK-3、Pax-8基因的表达后,探索Smad1/5/8在其中所起的作用。方法:将H9C2(2-1)大鼠胚胎心肌细胞分为4组:针对Pax-8基因的小干扰RNA(Pax-8 siRNA)组、ALK-3基因的小干扰RNA(ALK-3 siRNA)组、阴性对照(NC siRNA)组和空白对照组。前3组分别给予相应siRNA(5.0μL)和Lipofectamine 2000(5.0μL)配制成的siRNA-脂质体复合物溶液,空白对照组给予等量的细胞培养基。采用qRT-PCR测定Pax-8和ALK-3的mRNA表达,Western blot法检测磷酸化Smad1/5/8(p-Smad1/5/8)和半胱天冬酶(Caspase)-3表达。结果:与NC siRNA组和空白对照组比较,Pax-8 siRNA组的Pax-8 mRNA表达分别下调50%(P<0.05)和54%(P<0.05),ALK-3 siRNA组的ALK-3 mRNA表达分别下调49%(P<0.05)和53%(P<0.05),而NC siRNA组与空白对照组间差异无统计意义(P>0.05)。与NC siRNA组和空白对照组比较,ALK-3 siRNA组p-Smad1/5/8的蛋白表达量分别下调58%(P<0.05)和55%(P<0.05),Pax-8 siRNA组p-Smad1/5/8的蛋白表达量差异无统计意义(P>0.05)。NC siRNA组与空白对照组间差异无统计意义(P>0.05)。与NC siRNA组和空白对照组比较,Pax-8 siRNA组Caspase-3蛋白表达量分别上调76%(P<0.05)和81%(P<0.05),ALK-3 siRNA组Caspase-3的蛋白表达量分别上调135%(P<0.05)和140%(P<0.05),而NC siRNA组与空白对照组间差异无统计意义(P>0.05)。结论:在大鼠胚胎心肌细胞株中对基因ALK-3、Pax-8干扰后能引起细胞凋亡蛋白Caspase-3明显增多,并且在ALK-3 siRNA组发现Smad1/5/8蛋白磷酸化减少,而Pax-8 siRNA组中未发现Smad1/5/8磷酸化减少,提示Smad1/5/8在基因ALK-3被干扰后引起细胞凋亡蛋白Caspase-3增多中起重要作用。
Objective: To explore the effect of Smad1/5/8 by down-regulating selectively the level of expression of paired box gene8(Pax-8) or bone morphogenetic protein receptor type IA(BMPR-IA, also named ALK-3) in rat myocytes by RNA interference. Methods: The primary cultured H9C2(2-1) myocytes were divided into four groups: short interference RNA targeting Pax-8(Pax-8 siRNA) group, short interference RNA targeting ALK-3(ALK-3 siRNA) group, non-specifi c siRNA group as the negative control(NC siRNA), and the blank control group. The former three groups were treated with siRNA-liposome compounds consisting of siRNA(5.0 μL) and Lipofectamine 2000(5.0 μL), and the blank control group was treated with equal volume of culture medium. qRT-PCR was used to analyze the level of expression of Pax-8 mRNA and ALK-3 mRNA. Western blot was used to analyze the level of expression of phosphorylation protein Smad1/5/8 and Caspase-3. Results: In comparison with NC siRNA group and blank control group, the level of expression of Pax-8 mRNA in Pax-8 siRNA group was downregulated 50% and 54% respectively(both P<0.05), the level of expression of ALK-3 mRNA in ALK-3 siRNA group was downregulated 49% and 53% respectively(both P<0.05), while no signifi cant difference was found between the NC siRNA group and blank control group. After the transfection, the level of expression of p-Smad1/5/8 protein in ALK-3 siRNA group was signifi cantly lower than the level of the blank group(P<0.05) and NC siRNA group(P<0.05), while there was no signifi cantly difference between the blank group and NC siRNA group. The level of expression of Caspase-3 protein in the Pax-8 siRNA group and ALK-3 siRNA group was signifi cantly higher than the level of the blank group(P<0.05) and NC siRNA group(P<0.05), while there was no signifi cantly difference between the blank group and NC siRNA group. Conclusion: To down-regulate the level of expression of Pax-8 mRNA or ALK-3 mRNA may promote apoptosis protein Caspase-3 in myocardial cells. To down-regulate the level of expression ALK-3 mRNA can reduce Smad1/5/8 protein phosphorylation, suggesting Smad1/5/8 plays an important role after the gene causing interference in ALK-3 in increasing apoptotic protein Caspase-3.
Objective: To explore the effect of Smad1/5/8 by down-regulating selectively the level of expression of paired box gene8(Pax-8) or bone morphogenetic protein receptor type IA(BMPR-IA, also named ALK-3) in rat myocytes by RNA interference. Methods: The primary cultured H9C2(2-1) myocytes were divided into four groups: short interference RNA targeting Pax-8(Pax-8 siRNA) group, short interference RNA targeting ALK-3(ALK-3 siRNA) group, non-specifi c siRNA group as the negative control(NC siRNA), and the blank control group. The former three groups were treated with siRNA-liposome compounds consisting of siRNA(5.0 μL) and Lipofectamine 2000(5.0 μL), and the blank control group was treated with equal volume of culture medium. qRT-PCR was used to analyze the level of expression of Pax-8 mRNA and ALK-3 mRNA. Western blot was used to analyze the level of expression of phosphorylation protein Smad1/5/8 and Caspase-3. Results: In comparison with NC siRNA group and blank control group, the level of expression of Pax-8 mRNA in Pax-8 siRNA group was downregulated 50% and 54% respectively(both P<0.05), the level of expression of ALK-3 mRNA in ALK-3 siRNA group was downregulated 49% and 53% respectively(both P<0.05), while no signifi cant difference was found between the NC siRNA group and blank control group. After the transfection, the level of expression of p-Smad1/5/8 protein in ALK-3 siRNA group was signifi cantly lower than the level of the blank group(P<0.05) and NC siRNA group(P<0.05), while there was no signifi cantly difference between the blank group and NC siRNA group. The level of expression of Caspase-3 protein in the Pax-8 siRNA group and ALK-3 siRNA group was signifi cantly higher than the level of the blank group(P<0.05) and NC siRNA group(P<0.05), while there was no signifi cantly difference between the blank group and NC siRNA group. Conclusion: To down-regulate the level of expression of Pax-8 mRNA or ALK-3 mRNA may promote apoptosis protein Caspase-3 in myocardial cells. To down-regulate the level of expression ALK-3 mRNA can reduce Smad1/5/8 protein phosphorylation, suggesting Smad1/5/8 plays an important role after the gene causing interference in ALK-3 in increasing apoptotic protein Caspase-3.
引文
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[14]高瞻,来丹丹,张敏,等.Pax-8基因在大鼠心肌细胞凋亡中的作用[J].解放军医学杂志,2009,34(9):1082-1084.
[2]Ten Dijke P,Goumans MJ,Itoh F,et al.Regulation of cell proliferation by Smad proteins[J].J Cell Physiol,2002,191(1):1-16.
[3]Whitman M.Smads and early developmental signaling by the TGF-beta superfamily[J].Genes Dev,1998,12(16):2245-2262.
[4]Shi Y,Massague J.Mechanisms of TGF-beta Signaling from Cell Membrane to the Nucleus[J].Cell,2003,113(6):685-700.
[5]Kingsley DM.The TGF-beta superfamily:new members,new receptors and new genetic tests of function in different organisms[J].Genes Dev,1994,8(2):133-146.
[6]Li Z,Fei T,Zhang J,et al.BMP4 signaling acts via dualspecifi city phosphatase 9 to control ERK activity in mouse embryonic stem cells[J].Cell stem cell,2012,10(2):171-182.
[7]Daly AC,Randall RA,Hill CS.Transforming growth factor beta-induced Smad1/5 phosphorylation in epithelial cells is mediated by novel receptor complexes and is essential for anchorage-independent growth[J].Mol Cell Biol,2008,28(22):6889-6902.
[8]Wrighton KH,Lin X,Yu PB,et al.TGF-beta can stimulate Smad1 phosphorylation independently of BMP receptors[J].J Biol Chem,2009,284(15):9755-9763.
[9]Nicklas D,Saiz L.Computational modelling of Smad-mediated negative feedback and crosstalk in the TGF-beta superfamily network[J].J R Soc Interface,2013,10(86):201-363.
[10]Schlange T,Andree B,Arnold HH,et al.BMP2 is required for early heart development during a distinct time period[J].Mech Dev,2000,91(1-2):259-270.
[11]Monzen K,Shiojima I,Hiroi Y,et al.Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activate protein kinase kinase kinase TAK 1 and cardiac transcription factors Csx/NKx-2.5 and GATA-4[J].Mol Cell Biol,1999,19(10):7096-7105.
[12]Beppu H,Kawabata M,Hamamoto T,et al.BMP type II receptor is required for gastrulation and early development of mouse embryos[J].Dev Biol,2000,221(1):249-258.
[13]倪秋明,章佳颖,来丹丹,等.Pax-8基因在心脏发育中蛋白表达下调[J].温州医学院学报,2009,39(1):1-4.
[14]高瞻,来丹丹,张敏,等.Pax-8基因在大鼠心肌细胞凋亡中的作用[J].解放军医学杂志,2009,34(9):1082-1084.