Silence Of VEGFR2 Expression Mediated By PEI/siRNA Complexes

RNA interference (RNAi) is a natural phenomenon in the organisms, where 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 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 targeting RNA and become new long double-stranded RNA molecule. Then, 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 much stronger and more specific in gene silencing. So the technology of RNAi has rapidly been applied for disease treatment research by lots of domestic or foreign research institutions. Any disease-related genes overexpressed or mutated in vivo is a potential target.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 for cancer treatment. However, 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, successful delivery of siRNA in vivo needs carrier. However, 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, which makes it used more widely. One of the most commonly used cationic polymer is polyethyleneimine(PEI). The positively charged amino group of PEI and the negatively charged DNA phosphate groups integrated with each other by electrostatic absorption to form a stable nano-nucleic acid complexes which then integrated with the negatively charged cell membranes to enter cells with high transfection efficiency and targeting.In order to benefit the transfection for application of siRNA in vivo, we used Cationic polymer PEI to encapsulate siRNA molecules and prepared the siVEGFR2/PEI complexes, then transfected MS1 cells with siVEGFR2/PEI. Gene silencing efficiency in cells was valued with RT-PCR and westen blot. Moreover, the gene silencing efficiency and anti-tumor effect in vivo of siVEGFR2/PEI complexes delivered by intratumor injection or systemic injection were performed. All the tests here will lay the basis for the following modification of siVEGFR2 with tumor targeting molecules, and to optimize the delivery system for its application in vivo in the future.Objectives:1. To study the feasibility of PEI is used as drug delivery vector for siRNA in vitro and in vivo.2. To compared tumor inhibition effect of siVEGFR2/PEI deliveried through intratumor injection or i.v..Methods:1. Prepare the siRNA/PEI or siRNA/Lipofectamine2000 complexes. These complexes were used to transfect MSI cell, and there was PEI alone in blank group. Confocal microscopy was used to image subcellular distribution of the CY3-labeled siRNA in MS1 cells.2. Transfect MS1 cells. In control group and siVEGFR2-transfected group, MS1 cells were transfected with scrambled control siRNA and siRNA targeting mouse VEGFR2 respectively, and in blank group, there was no siRNA transfection. At last, the knockdown of VEGFR2 expression in MS1 cells was determinated by semi-quantitative RT-PCR and western blot. One-way ANOVA statistic method was used to evaluate whether there was a significantly difference for VEGFR2 mRNA and protein level among each transfection group.3. Inoculate tumor in vivo. Subcutaneous tumors were induced by inoculation of 1×106 A549 cells in the flank of the mice. As the tumor volume of≈60-70mm3, mice received each siRNA complexes respectively by intratumor injection of a solution of 0.05ml with 0.5nmol siRNA or i.v. injection of a solution of 0.15ml with 1.5nmol siRNA via the tail vein. The experiment is divided into five groups and each group has five mouse.1):saline,2):siRNA/PEI,3):siVEGFR2/Lipofectamine2000,4): siVEGFR2/PEI,5):siVEGFR2/PEI(vein). Tumor size was measured every two days using calipers, and tumor volume was calculated using the formula:1/2×a×b2, where a and b represent the larger and smaller tumor diameters respectively. Single-factor repeated measure variance analysis method was applied to compare whether there was a significant difference among each group for the tumor volum and tumor-bearing mice weight.4. Analysis the gene silence efficiency. Two days after final injection, the tumor-bearing mice were sacrificed. Semi-quantitative RT-PCR and Western blot methods were proceeded to test the RNAi effect in vivo. One-way ANOVA statistic method was used to evaluate whether there was a significantly difference among each group for VEGFR2 mRNA and protein level.5. Perform the mechanism of tumor inhibition. Two days after final injection, the tumor-bearing mice were sacrificed for immunohistochemical staining. Tumor sections were incubated with anti-mouse CD31 antibody to assess the intratumoral microvessel density.6. Analysis the immunogenesis. Cytokines derived from mouse serum were determinated by Bio-plex system. One-way ANOVA statistic method was used to evaluate whether there were significant differences among each group for cytokines.Results:1. Substantial fluorescence present in cytoplasm as well as in the nucleus for lipofectamine 2000-transfected cells. By contrast, PEI-transfected cells exhibited much fluorescence in cytoplasm with no evidence of nuclear accumulation.2.48hs After transfection of MS1 cells with siVEGFR2/lipofectamine2000 or siVEGFR2/PEI, the total mRNA from MS1 cells were extracted by Trizol reagent and measured the VEGFR2 gene expression by semi-quantitative RT-PCR. The grey scale ratios of VEGFR2 band and (3-actin band measured by Imge J software were quantitied as the index for the silence of the VEGFR2 mRNA expression in MS1 cells. Statistically significant differences were observed among the four groups by one-way ANOVA analysis (F=58.345, P=0.000), Further statistical analysis (by LSD) indicated that the VEGFR2 mRNA levels between siVEGFR2/Lipofectamine2000 group or siVEGFR2/PEI group and blank group were significant different (P=0.000), but there was no significant difference between blank group and control group (p=0.579) or siVEGFR2/Lipofectamine2000 group and siVEGFR2/PEI group (P=0.368).3.48hs after transfection of MS1 cells with siVEGFR2/lipofectamine2000 or siVEGFR2/PEI, the total protein of MS1 cells were extracted by RIPA reagent. The level of VEGFR2 protein expression was measured by western blot. The grey scale ratio of VEGFR2 band and GAPDH band measured by ImgeJ software were quantitied as the index for the knock-down of VEGFR2 protein expression in MS1 cells. Statistically significant difference was observed among the four groups by one-way ANOVA analysis (F=22.191, P=0.000). Further statistical analysis indicated that the VEGFR2 protein levels between siVEGFR2/Lipofectamine2000 group or siVEGFR2/PEI group and blank group were significant down regulated (P=0.000), but there was no significant difference between blank group and control group (p=0.476) or siVEGFR2/Lipofectamine2000 group and siVEGFR2/PEI group (P=0.763)3. Subcutaneous tumors were induced by inoculation of A549 cells in the flank of mice. Tumor size and tumor-bearing mice weight were measured every two days, Single-factor repeated measure variance analysis method was applied to compare the tumor volum and tumor-bearing mice weight change among each group, There was no significant difference for tumor-bearing mice weight(F=0.801,P=0.539) but tumor volumn(F=23.559, P=0.000). Multiple comparisons:as compared to saline group, there was no statistic difference of tumor volumn for siRNA/PEI and siVEGFR2/PEI (v) group (P=0.202 and P=0.055 respectively), but siVEGFR2/Lipofectamine2000 and siVEGFR2/PEI group (P=0.000).4. Two days after the last injection, the total RNA of tumors were extracted by Trizol reagent. The VEGFR2 gene expression was measured by semi-quantitative RT-PCR. Statistically significant difference was observed among the five groups of VEGFR2 mRNA by one-way ANOVA analysis (F=40.090, P=0.000), Further statistical analysis (by LSD) indicated that siVEGFR2/Lipofectamine2000 or siVEGFR2/PEI group VEGFR2 mRNA levels were significant different as compared to that in saline group (P=0.000), but there was no significant difference between siRNA/PEI or siVEGFR2/PEI (v) and saline group (P=0.185 and P=0.171 respectively).5. Two days after the last injection, the total protein of tumors were extracted by RIPA reagent and the VEGFR2 protein expression was measured by Western blot. Statistically significant difference was observed in VEGFR2 protein level among the five groups by one-way ANOVA analysis (F=23.572, P=0.000), Further statistical analysis (by LSD) indicated that siVEGFR2/Lipofectamine2000 or siVEGFR2/PEI group VEGFR2 protein levels were significant different as compared to that in saline group (P=0.000). But there was no significant difference between siRNA/PEI or siVEGFR2/PEI (v) and saline group (P=0.323 and P=0.260 respectively).6. Tumors were harvested, sectioned and stained with CD31 antibody to perform the Immunohistochemical staining. The microvessles in tumor section in siVEGFR2/Lipofectamine2000 group and siVEGFR2/PEl group were less than that in the other groups.7. Cytokines were determination by Bio-Plex system. The results displayed that, as compared to that in saline group, there were significant differences of IL-10 level in siVEGFR2/PEI (v) group(P=0.021), IL-4 level in siRNA/PEI group(P=0.024) and IL-1βlevel in siVEGFR2/PEI group (P=0.015).Conclusion:1. PEI and Lipofectamine2000 could effectively delivery siRNA molecules into the RNAi pathway in cytoplasm.2. siVEGFR2 mediated by PEI or Lipofectamine2000 could effectively silence the VEGFR2 gene expression in MS1 cell.3. The anti-tumor effect of siVEGFR2/PEI by intratumor delivery was much stronger than that by the i.v. delivery way, which implied that the unmodified PEI had not tumor targeting. All the results here layed a solid foundation for further study to improve the drug delivery system.4. The antitumor effect of siVEGFR2/PEI via intratumor delivery is dependent on RNAi but immunostimulation induced by siRNA.5. siVEGFR2/PEI deliveried by intratumor injection method could block the VEGF/VEGFR2 signal pathway which is very important for new formed microvessles to growth and then could result in tumor inhibition, by specific gene silencing effect.6. It is feasible that PEI could be used as delivery vector for siRNA in vitro and in vivo.