The enhanced effect of nuclear localization signal peptide during the ultrasound targeted microbubbles destruction mediated gene transfection

Abstract

Objective To investigate the transfection efficiency combining ultrasound targeted microbubbles destruction (UTMD) and nuclear localization signal (NLS) peptide for facilitating the plasmid of enhanced green fluorescent protein (pEGFP) into nucleus. Methods This study was divided into 3 groups, group A: UTMD+ pEGFP; group B: UTMD+ NLS+ pEGFP; group C: Lipo3000+ pEGFP. The NLS was labeled by FITC and pEGFP was marked by Cy3. The different mole ratio was adjusted between NLS and pEGFP for observing the best ratio of combination.The human umbilical vein endothelial cells (HUVEC) were transfected by the optimum ultrasonic irradiation parameters and the optimal NLS/pEGFP mole ratio. Six hours after transfection, the rate of Cy3 labeled pDNA into cells and nuclear were detected by flow cytometer and laser confocal microscope respectively. Forty-eight hours after transfection, the transfection efficiency was detected by flow cytometer; the survival rate of cells was measured by CCK8. RT-PCR and Western technology were used to detect the relative expression amount of mRNA and protein. The above indicators were compared among 3 groups, which were used to evaluate the enhanced effect of NLS in UTMD mediated gene transfection. Results ①Six hours after transfection, the NLS with green fluorescence and pEGFP with red fluorescence can show at the same site and signal intensity within the cell, that suggested a combination between them, agarose gel electrophoresis showed that the best molar ratio of NLS/pEGFP combining was 104∶1. ②Six hours after transfection, the rates of pEGFP into the cells were (63±12)%, (80±10)% and (92±8)%; the rates of pEGFP into the nucleus were (17±3)%, (50±12)% and (35±8)% in 3 groups respectively(P 80% in all groups; the transfection efficiency, relative quantity of mRNA and protein expression were increased gradually. There were significant differences among 3 groups(P<0.05). They were 1.6, 2.3 and 2.4 times in group B than those in group A, still lower than those in group C. Conclusions The UTMD combining NLS can promote the pEGFP into nucleus for improving the transfection efficiency. The NLS peptide can play an enhanced effect as a new strategy of UTMD. Key words: Sonication; Microbubbles; Transfection; Nuclear localization signal