ALK-3、Pax-8基因抑制后大鼠心肌细胞中Smad1/5/8的表达

Expression of Smad1/5/8 gene in the rat myocardium after the inhibition of ALK-3 gene and Pax-8 gene

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 ex-pression 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 di-vided into four groups： short interference RNA targeting Pax-8 （Pax-8 siRNA） group, short interference RNA targeting ALK-3 （ALK-3 siRNA） group, non-speciifc 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 （bothP〈0.05）, the level of expression of ALK-3 mRNA in ALK-3 siRNA group was downregulated 49% and 53% respectively （bothP〈0.05）, while no＆amp;nbsp;signiifcant 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 signiifcantly lower than the level of the blank group （P〈0.05） and NC siRNA group （P〈0.05）, while there was no signiifcantly 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 signiifcantly higher than the level of the blank group （P〈0.05） and NC siRNA group （P〈0.05）, while there was no signiifcantly difference between the blank group and NC siRNA group.Conclu-sion： 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.