Effect Of Silica Nanoparticle-mediated MicroRNA-24 Transfection On Cardiomyocyte Apoptosis

ObjectiveFunctionalization of silica?Silica?surface by polyethyleneimine?PEI?to form F-Silica,and then combined with miR-24 by electrostatic adsorption to form gene carrier complex F-Silica-miR-24.The toxicity and transfection efficiency of neonatal rat ventricular myocytes?NRVM?were evaluated in an in vitro study,and the optimal ratio was selected.The effects of the proapoptotic protein Bim and cardiomyocyte apoptosis after oxidative stress injury were further studied.Methods1)F-Silica was constructed,and the microstructure,particle size and carrying charge of the material were characterized by transmission electron microscopy,nano-particle size and Zeta potential analyzer.2)The biocompatibility of F-Silica was evaluated by CCK-8/live-dead staining,etc.The optimal ratio of the gene complex vector F-Silica-miR-24 was screened by agarose gel electrophoresis.3)Cell uptake studies evaluate the transfection efficiency of the F-Silica-miR-24.4)The experiment was divided into 6 groups:blank control group?NC group?was simply added with 100?l PBS;H₂O₂ group was simply added with final concentration of 100?mol/L H₂O₂ for24 h;miR-24 mimics?miR-24-mimics?Group;miR-24 mimics control?miR-24-mimics NC?group;miR-24 inhibitor?miR-24-inhibitor?group;miR-24inhibitor control?miR-24-inhibitor NC?group were pre-transfected with F-Silica-loaded 200 nM oligonucleotides for 24 h prior to H₂O₂ treatment.After in vitro transfection,the effects of the target gene and proapoptotic protein Bim on neonatal rat ventricular myocytes were evaluated by real-time PCR and Western blotting.TUNEL staining was used to further evaluate the effect of cardiomyocyte apoptosis.Results1)The size of the synthesized Silica was concentrated between 40-60 nm;when Silica and DSP-PEI were constructed with the mass ratio of 1:1 to construct the gene vector F-silica,the potential was about+21 mv.2)When the concentration of F-Silica was0.25 mg/ml,200 nM of miR-24 was completely loaded as the final concentration of F-Silica in the F-Silica-miR-24 group.And when co-cultured with cardiomyocytes at a dose twice the concentration required for loading miRNA,there was no significant effect on the viability of cardiomyocytes.3)Cellular uptake studies clearly demonstrate that F-Silica-miRNA complex nanocarriers can effectively deliver miRNA molecules into cardiomyocytes,and the transfection efficiency can reach over 80%.4)After treatment with 100uM H₂O₂ for 24h,the survival rate of cardiomyocytes was about 50%,and the model of myocardial apoptosis was successfully modeled.The results of real-time PCR showed that compared with the NC group,the expression of miR-24 in the H₂O₂ group,miR-24 mimics NC group and miR-24 inhibitor NC group decreased,the difference was statistically significant?P<0.05?.The expression of miR-24 was significantly increased in the miR-24 mimics group,and the expression of miR-24 was significantly decreased in the miR-24 inhibitor group?P<0.05?.Western blotting showed that compared with NC group,the expression of protein Bim in H₂O₂group and miR-24 inhibitor group was significantly increased?P<0.05?,however,there was no significant difference in the expression of protein Bim between NC group and miR-24 mimics group?P>0.05?.TUNEL staining showed that overexpression of miR-24 in vitro inhibited cardiomyocyte apoptosis and promoted cardiomyocyte survival;inhibition of miR-24 expression significantly increased the incidence of cardiomyocyte apoptosis.Conclusion1)Using PEI800 as a surface modification of silica,the nano-gene carrier F-Silica produced by the synthesized Silica after DSP-PEI functionalization?W/W 1:1?is not only has good biocompatibility,but also has better gene transfection ability.2)Compared with the H₂O₂ group,the expression level of miR-24 in the miR-24 mimics group was significantly increased,and the expression of the proapoptotic protein Bim was significantly inhibited.3)F-Silica-mediated miR-24 transfection can significantly inhibit cardiomyocyte apoptosis induced by oxidative stress injury and promote myocardial cell survival.