ShRNA-mediated Gene Silencing Of IGF-IR Prevented Cardiac Hypertrophy In Murine Model

ObjectiveThat polymer potentiating gene transfection into target cells has been evaluated in various cancer cell lines. But the feasibility of polymer-based gene transfection to cardiac muscle cells has never been systematically evaluated. IGF1/IGF-1R signal pathway which activated by hemodynamic mechanical stress in heart mediates smooth muscle cell hypertrophy. In this study, we evaluated the transfection efficiency and therapeutic potential of polymer-based transfection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-IRs) in cardiac muscle cells and heart.Methods:Two siRNA expression vectors, pIGF-1R-siRNAl, pIGF-1R-siRNA2 targeting IGF-1R, were constructed using pENTR/U6 vector, and a vector targeting luciferase gene, pcontrol-siRNA was constructed as control. The levels of mRNA and protein expressions were determined by RT-PCR and Western blot analysis after recombinant plasmid pIGF-1R-siRNA1, pIGF-1R-siRNA2 and pcontrol-siRNA were transfected into cardiac cells 48 h respectively. Cells grown from suckling mice. Plasmids expressing GFP and shRNA targeting IGF-1R (pGFPshIGF-1R) were constructed by corporation of Genepharma. Transfection in vitro was performed as the manufacturer’s protocol in a 6-well plate. In vivo transfection, a total of 0.2mL MegaTran 1.0/DNA mixture was instilled into the isolated external jugular vein segment through internal jugular vein. The cells and mice received a continuous infusion of norepinephrine induced hypertrophy.IGF-1R was detected by RT-PCR and western blot, ANP and β-MHC detected by RT-PCR and transthoracic echocardiography was performed in awaked mice in order to evaluate the hypertrophy of heart. After 18 days, mice were sacrificed and the hearts were fixed with 10% formaldehyde solution, embedded in paraffin. Tissue sections were stained and morphometric analysis was performed.ResultsAfter 48 h transfection, level of IGF-1R mRNA and protein were significantly decreased in IGF-1R-siRNA2-cardiac cells (47±1.8% and 41±1.2% respectively). Similar but less profound knockdown of IGF-1R expression at both mRNA and protein levels were induced by pIGF-1R-siRNA1.Polymer-based transfection provided a high efficiency in transgene expression in vitro. In vivo, the delivering efficiency of the pGFPshIGF-1R plasmids into the cells was significantly high. In vivo, IGF-1R specific-shRNA inhibited IGF-1R protein by 72.2±6.8%,80.7 ±9.6% and 84.5±5.6% at 24h,48h, and 72 h, respectively. Norepinephrine induced cardiac hypertrophy and increased the level of IGF-1R, ANP and p-MHC, reduced the heart function. Polymer-based delivering sh-IGFIR inhibited the cardiac hypertrophy, reduced HW: BWs (heart weight-to-body weights), improved the heart function compared with controls. And these effects were mediated via inhibiting PI3K/AKT pathway.ConclusionsOur findings indicated that polymer-based transfection may be a promising method that allows the targeting of gene therapy to heart failure.