SARA低表达对Activin A/Smads环路活性的影响

Effect of SARA suppression on the activity of Activin A/Smads signal loop

目的探讨下调Smad锚捉蛋白(SARA)表达对Activin A/Smads环路活性的影响。方法利用RNA干扰技术,设计靶向大鼠SARA基因的短发卡RNA(shRNA),瞬时转染激活素A(ActA)高表达的大鼠嗜铬细胞瘤PC12细胞。转染48h后倒置荧光显微镜观察转染效率,实时定量PCR、Western blot检测SARA基因的表达情况。对阳性转染组及阴性转染组细胞换液3h后,Western blot检测Smad3总蛋白及磷酸化蛋白的表达。结果靶向SARA基因的SARA-shRNA及对照NC-shRNA质粒的转染效率均超过70%。转染48h后,SARA基因转录及蛋白水平的表达显著降低。细胞换液3h后,与NC-shRNA组比较SARA-shRNA组Smad3总蛋白及磷酸化蛋白下调44%和57%。结论 SARA基因低表达下调ActA/Smads环路活性。

Objective To explore the effect of Smad anchor for receptor activation(SARA)suppression on the activity of Activin A/Smads signal loop.Methods With the use of RNA interference(RNAi)technology,the short hairpin RNA(shRNA)plasmid targeted to rattus SARA gene was designed and synthetized.After transfected into Act A overexpressioned PC12 cells,the transfection efficiency was observed under a fluorescence microscope.Then the expressions of SARA mRNA and protein were detected 48 hafter transfection by Real-Time RT-PCT and Western blot.The effect of SARA suppression on Smad3 expression and phosphorylation was tested by Western blot,after 3 hof media exchange.Results After 48 hof shRNA plasmid transfection,the transfection efficacies were observed under a fluorescence microscope,which was more than 70%in both SARA-shRNA group and NC-shRNA group.The delivery of pGenesil-3-SARA-shRNA(SARA-shRNA group)resulted in a dramatically knockdown of SARA expression.After 3 h of media exchange,the expressions of total and phosphorylated Smad3 decreased by 44%and 57%in SARA-shRNA group,compared to that in NC-shRNA group.Conclusion Suppression of SARA down regulated the activity of ActA/Smads loop signal.