Development And Functional Verification Of MiR-16 Tumor Targeting Nano Drug Delivery System

In recent years,more and more miRNA molecules have been confirmed to have the ability to regulate the occurrence and development of tumors,but the application of miRNA molecules in clinical tumor therapy is still limited by the incomplete drug delivery system of nucleic acid molecules and the regulatory mechanism of miRNA molecules on tumor cells.The purpose of this study is to broaden the pathway of nucleic acid drug molecule delivery,and to clarify the possibility of miRNA molecule application in tumor therapy.In this study,the cRGD-R9-SS-R9 peptide was designed and synthesized to encapsulate miR-16,forming the nanoparticle molecules.The biological functions of the nanoparticle were investigated in vitro,the effect and mechanism of inhibiting the growth of ovarian cancer cells and reversing cisplatin resistance were verified,and the application possibility of the tumor targeting nano drug delivery system.Methods1.Determine the research objects of miRNA molecules,synthesis and characterization of polypeptides and nano-particle molecules.The literatures were reviewed and flow cytometry was used to screen and verify to obtained miRNA molecules that promoted the apoptosis of ovarian cancer cells SKOV-3 cells and enhanced their cisplatin sensitivity.R9-SS-R9 and cRGD-R9-SS-R9 peptides were synthesized,and their structures and purity were determined by high performance liquid chromatography and mass spectrometry.Nanoparticles were synthesized encapsulating miRNA with peptide/miRNA weight ratios of 5:1,10:1,20:1 and 40:1.2.Characterization of nanoparticle moleculesAgarose gel electrophoresis,Malvin instrument and TEM were used to characterize the particle size,PDI,potential,morphology and stability of the nanoparticles,and to determine the optimal peptide/miRNA weight ratio.The cytotoxicity test of nano-particle materials was completed by CCK8 method.3.Efficiency verification of nanoparticles(1)Efficiency of nanoparticle uptake:R9-SS-R9/siNC-cy5,cRGD-R9-SSR9/siNC-cy5 and siNC-cy5 at 200nM and 400nM were transfected in SK-OV-3 cells,and the uptake of nanoparticle by cells was detected by flow cytometry.(2)Verification of transfection efficiency at RNA level:Stem loop qRT-PCR was used to detect the relative expression level of miR-16 in cells after transfection of R9SS-R9/miRNA and cRGD-R9-SS-R9/miRNA nanoparticles.(3)The distribution of nanoparticles in cells:the co-location map of R9-SS-R9/siNC-cy5 and cRGD-R9-SS-R9/siNC-cy5 nanoparticles and lysosomes was captured by using the confocal high-content imaging system to observe whether the nanoparticles escape from the internal circulation pathway.4.To evaluate in vitro the effects of nanoparticles on the anti-proliferation and reversal of cisplatin resistance of ovarian cancer cells(1)Pro-apoptosis effect of nanoparticles on cells:R9-SS-R9/miRNA and cRGDR9-SS-R9/miRNA nanoparticles were transfected intracellular,and cisplatin supplementing group was set,and cell apoptosis was detected by flow cytometry.(2)The mechanism affecting apoptosis and cisplatin sensitivity of cells was studied at the protein level:R9-SS-R9/miRNA and cRGD-R9-SS-R9/miRNA nanoparticles were transfected in cells,and the protein expression changes of Bcl-2 and Chk-1 in cells were detected by Western blot.Results1.cRGD-R9-SS-R9 polypeptide was successfully synthesized and the appropriate weight ratio was found out to encapsulate miR-16 to form nanoparticle with good morphology and stable structure.2.CCK8 method was used to verify that cRGD-R9-SS-R9 polypeptide had low toxicity.3.Efficiency verification of nanoparticles.(1)Flow cytometry was used to verify that the intake of cRGD-R9-SS-R9/siNCCy5 nanoparticles was higher than that of the control group.(2)The expression level of miR-16 after SK-OV-3 cells were transfected with cRGD-R9-SS-R9/miR-16 nanoparticles was significantly higher than that of cRGDR9-SS-R9/siNC nanoparticles by stem loop RT-qPCR(p<0.01).(3)Confocal high-content imaging system was used to verify that cRGD-R9-SSR9/miR-16 nanoparticles could be well ingested by SK-OV-3 cells and distributed in the cytoplasm.4.To evaluate in vitro the effects of nanoparticles on the anti-proliferation and reversal of cisplatin resistance of ovarian cancer cells.(1)The pro-apoptotic effect of cRGD-R9-SS-R9/miR-16 nanoparticles on SKOV-3 cells was significantly higher than that of the control group(p<0.01),the apoptosis rate of cRGD-R9-SS-R9/miR-16 nanoparticles combined with cisplatin was significantly higher than that of the control group(p<0.0001).(2)Western blot assay was used to verify that the protein levels of Bcl2 and Chk1 in the experimental group were lower than those in the control group(p<0.01).ConclusioncRGD-R9-ss-R9 polypeptide encapsulates miR-16 to form nanoparticles,which possess stable structure with low cytotoxicity,and enhance the uptake of miR-16 and endosomal release,and promote the apoptosis of cisplatin resistant SK-OV-3 cells and increase their sensitivity to cisplatin by reducing the expression of Bcl-2 and Chk-1,realizing miR-16 tumor targeted delivery.The results laid a foundation for the application of miR-16 in vivo.