Serum Stability Of SiRNA Targeting VEGFR2 And Its Gene Silencing Efficiency

RNA interference is a natural phenomenon in the organisms,its mechanism is: exogenous or endogenous long double-stranded RNA is cleaved into short 21 to 23 nucleotide fragments known as small interfering RNA(siRNA) by an RNA enzyme, Dicer.These so-called small interfering RNAs(siRNAs) are subsequently incorporated into a nuclease-containing multiprotein complex called the RNA-induced silencing complex(RISC),which is then activated through unwinding of the double-stranded siRNA.The sense strand is degraded and then the activated RISC is guided by the antisense strand to bind its complementary target RNA and become new long double-stranded RNA molecule.After that,the newly formed long double-stranded RNA molecule is degraded by Dicer into new siRNA fragments again,which can enter a new round of gene silencing cycle,and therefore the target mRNA is destroyed.Compared with the antisense oligonucleotide and ribozyme,the siRNA has no immunogenicity and stronger and more specific gene silencing efficiency.So the technology of RNAi has rapidly been applied for disease treatment research by lots of domestic or foreign research institutions,any disease-related gene which expression can lead to disease is potential target.But as the effector of RNAi, siRNA has the dual characteristics of nucleic acid and micromolecule compounds. When applied in vivo as gene drug,siRNA molecules are susceptible to nuclease and have poor stability,short half-life,low transfection efficiency,poor targeting and so on.So the key to the application of siRNA molecules,is how to overcome the cellular membrane barrier,and makes siRNA molecules enter into the RNAi pathway in cytoplasm.Currently,most siRNA administration need carrier,but most of these carriers have more or less problems such as poor targeting,toxic immune responses,low transfection efficiency and so on.Although with a high efficiency of gene transfection,viral vector has a potential carcinogenicity and immunogenicity; Cationic liposome has a larger cell toxicity and instability;Cationic polymer has lower immunogenicity and cell toxicity,but its low transfection efficiency and complicated design hinder its clinical application.In recent years,it is reported that several chemical stability and tissue targeting modifications to the specific position of siRNA have not affected its gene-silencing activity,and thus has a possibility to overcome the disadvantage of the current administration using vector and become an ideal method for siRNA delivery.vascular endothelial growth factor receptor-2(VEGFR2) is one kind of tyrosine kinase receptor which contains three parts:an extracellular domain,a transmembrane domain and an intracellular tyrosine kinase structure domain.VEGFR2 binds to VEGF and then activates the VEGF/VEGFR2 pathway,which plays an important role in angiogenesis.Therefore,designing siRNA molecule targeted against VEGFR2 gene to silence VEGFR2 gene expression and block VEGF/VEGFR2 pathway can inhibit tumor angiogenesis and thus has the application prospect of cancer treatment.In order to explore the feasibility of tissue targeting modification for siRNA administration,we designed and synthesized siRNA molecule targeting mouse VEGFR2 gene,studied on its serum stability and gene silencing efficiency,which may provide theoretical basis for the following tissue targeting modifications and application in vivo. Objectives:1.To design siRNA targeting mouse VEGFR2 and study on its serum stability.2.To study the gene silencing efficiency of the designed siRNA.Methods:1.Based on the mRNA sequences of mouse VEGFR2 gene published in GeneBank and combined the method provided by Ambion's siRNA Target Finder and literatures,the siRNA targeted against VEGFR2 were designed.Then the designed siRNA were synthesized and diluted in RNase free water,and then were mixed in a 1:1 ratio with fresh serum to give 50%serum concentration and incubated at 37℃.Gel electrophoresis was performed to visualize the intact siRNA from different incubation times.2.The Oligofectamine 2000 were used to mediate unmodified siRNA to transfect MS1 cell.In control group and siVEGFR2 group,MS1 cell were transfected with control siRNA and siRNA targeting mouse VEGFR2 respectively,and in blank group,there were no siRNA transfection.At last,the knockdown of VEGFR2 expression in MS1 cell were evaluated by semi-quantitative RT-PCR and western blot.Results:1.Based on the mRNA sequences of mouse VEGFR2 gene published in GeneBank and combined the method provided by Ambion's siRNA Target Finder and literatures,we designed the target sequence for siRNA targeting mouse VEGFR2,which sequence was 5'-AAGCTCAGCACACAGAAAGAC-3'(seq No:NM₀10612.2),and the siRNA molecules targeting mouse VEGFR2,which sequence were as follows:Sense:5'-GCUCAGCACACAGAAAGACdTdT-3'; Antisense:5'-GUCUUUCUGUGUGCUGAGCdTdT-3'.2.The siRNA was diluted to 5μM with RNase free water,and then mixed with the same volume of fresh serum and incubated at 37℃.Gel electrophoresis was performed to visualize the intact siRNA from different incubation times.The results showed that naked siRNA incubated in serum was gradually degraded with extention of incubation and almost could not be detected after 24 hours.3.After liposome-mediated siRNA transfection into MS1 cell,we extracted the total mRNA of MS1 cells by Trizol reagent and measured the VEGFR2 gene expression by semi-quantitative RT-PCR.Calculating the grey scale ratio of VEGFR2 band andβ-actin band by ImageJ software as the index of VEGFR2 expression in MS1 cells,statistically significant difference was observed among the three groups by one-way ANOVA analysis(F=12.674,P＜0.01),Further statistical analysis(by LSD) indicated that the VEGFR2 mRNA levels between siVEGFR2 group and blank group or control group were significant difference (P＜0.01),but there was no significant difference between blank group and control group(p=0.618).4.After transfection of siRNA into MS1 cells by liposome-mediated way,we extracted the total protein of MS1 cells by RIPA reagent and measured the VEGFR2 protein expression by western blot.Calculating the grey scale ratio of VEGFR2 band andβ-actin band by ImageJ software as the index of VEGFR2 protein expression in the MS1 cells,statistically significant difference was observed among the three groups by one-way ANOVA analysis(F=300.457,P＜0.01).Further statistical analysis indicated that the VEGFR2 protein levels between siVEGFR2 group and blank group or control group were significant down regulated(P＜0.001),but there was no significant difference between blank group and control group(p=0.660).Conclusions:1.The stability of naked siRNA in serum is short and not suitable to apply directly in vivo.2.The designed siRNA can effectively silence the VEGFR2 gene expression.