Expression of Smad1/5/8 gene in the rat myocardium after the inhibition of ALK-3 gene and Pax-8 gene
1.Department of Cardiology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015; 2.Department of Cardiology, the Affiliated Hospital of Hangzhou Normal University, Hangzhou, 310015
Abstract：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-specific 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 significant 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 significantly lower than the level of the blank group (P<0.05) and NC siRNA group (P<0.05), while there was no significantly 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 significantly higher than the level of the blank group (P<0.05) and NC siRNA group (P<0.05), while there was no significantly 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.
