Ultrasound-mediated destruction of gene-loaded microbubbles enhanced the transfection of phospholamban antisense RNA into cardiac myocytes

Abstract

Objective To explore whether ultrasound-mediated microbubbles destruction could enhance anti-sense RNA transfection and expression. Methods Phospholamban antisense RNA eukaryon vector PcDNA 4. 1-asPLB was successfully constructed and it was transfected into cardiac myocytes by various methods including calcium phosphate precipitation, ultrasound exposure and ultrasound-mediated microbubbles destruction. The expression of PLB and sarcoplasmic retculum Ca2+ ATPase (SERCA2a) in cardiac myocytes was tested by RT-PCR and western blot. Results The transfection and expression of PcDNA 4. 1-asPLB increased significantly in cells treated with ultrasound-mediated microbubbles destruction compared to other transfer groups( P ＜0.05). The expression of PLB was inhibited specifically after cardiac myocytes were transfected with PcDNA 4. 1-asPLB. There was no change of PLB expression after cardiac myocytes transfected with PcDNA 4. 1 ( P ＜0.05). Though the expression of SERCA2a never exhibited any changes after PcDNA 4. 1-asPLB transfection, the PLB/SERCA2a ratio decreased markedly. Conclusions As a highly effective antisense RNA transfer method, ultrasound-mediated microbubbles destruction can enhance the transfection and expression of the PcDNA 4. 1-asPLB significantly. The PcDNA4. 1-asPLB transfection inhibits the expression of PLB and result in decrease of PLB/SERCA2a ratio in cardiac myocytes. Key words: Ultrasonography; Microbubbles; RNA,antisense; Transfection; Phospholamban