Small Interfering RNA (siRNA) Targeting VEGF Inhibits Initial A549 Tumor Growth In A Xenograft Murine Model And Angiogenesis Induced By A549 Cell On CAM Assays

Background:Antiangiogenic therapy has emerged as an important concept in the treatment of solid tumors, including non-small cell lung cancer (NSCLC). Vascular endothelial growth factor (VEGF) represents an important therapeutic target, as it is the primary mediator of angiogenesis and is induced by multiple tumor-relevant stimuli. Although antivascular strategy that targets the endothelial cells of the host was shown to be very efficient at reducing tumor growth and was not expected to trigger tumor resistance, recent studies have demonstrated that sustained high-level secretion of angiogenic stimulator from the tumor cells such as VEGF, can enable tumors to bypass antiangiogenic treatments. RNA interference (RNAi) is now being exploited as a powerful tool for reverse genetics, and shows great promise for therapeutic applications. Some authors developed vectors or viruses to produce RNAi which could be used in vivo experiments. However, the diffieultyof gene delivering still limits in vivo study. The aim of the present work was to determine whether blocking the ability of A549 cells to express endogenous VEGF by siRNA in a mouse xenograft experiments could affect the tumor growth and A549 cell induced angiogensis. Part 1 Small interfering RNA (siRNA) targeting VEGF inhibits initial A549 tumor growth in a xenograft murine model.Objective:To investigate the effect of VEGF siRNA on A549 cells in vitro using two transfection reagents and if it could inhibit A549 cell tumour growth in a xenograft murine modle.Materials and methods:Construction of siRNA vector: Three pairs of siRNAs were designed to downregulate human VEGF(GenBank Accession Number NM-003376) expression.Three pairs of VEGFsiRNA-plasmid and non-specific-plasmid were transfected into A549 cells through PEI or lipofectamine 2000, respectively. The most effective pair of VEGFsiRNA-plasmid was selected by ELISA and realtime RT-PCR. To generate A549 cell lines stably expressing VEGF siRNA by drug selection with G418. Transfection efficiency and toxicity were measured by fluorescence detection or MTT. A549 cells were injected in nude mice to gengerate xenograft than transfected with VEGF siRNA or co-transfected with PEI or lipo fectamine 2000, respectively. The stable A549 cell clone which expressesVEGFsiRNA-plasmid or non-specific-plasmid and A549 cell without transfection were injected in nude mice respectively, to evaluating their biologic effects.Results:The highest transfection efficiecy of PEI or . lipofectamine 2000 was 41% or48%. Cell survival rate with PEI or lipofectamine 2000 was similar as (65.4±4.3) %,(68.3±3.0) %. Transient-transfection of VEGFsiRNA-plasmid in A549 cell induced 28%,40%,30% reduction in the ratio of VEGF mRNA copies to hGAPDH mRNA copies, respectively.. Stable-transfectiong of VEGF siRNA plasmid induced 49%, 65%, 49%, reduction in the ratio of VEGF mRNA copies to hGAPDH mRNA copies, respectively. ELISA of VEGF revealed that tansient-transfection of VEGFsiRNA-plasmid in A549 cell induced slightly levels of reduction in VEGF secretion as compared with untransfected cells: VEGFsiRNAl induced 16% reduction in VEGF secretion, VEGFsiRNA2 induced 19%, VEGFsiRNA3 induced 17%. Stable-transfectiong of VEGF siRNA plasmid induce 32%,61%,42% reduction in VEGF secretion.After a certain period of injection with A549 cells into the axilla of nude mice, visible tumors had developed to be palpable at the injection sites. Transient-transfection of VEGFsiRNA-plasmid in tumour with or without PEI or lipofectamine 2000 showed on effect on tumour growth or VEGF in tumour content.Stable-transfection A549 cells of nude mice xenograft model showed tumour formation was 100%. Analysis of the growth curves show that VEGFsiRNA reduced tumor volume from day 45 after injection compared with non-silencing group or control group. The logarithmic regression analysis of the growth curves showed that VEGFsiRNA do not significantly reduce the growth rate of the tumors but induced a slight increased in VEGF siRNA group as compared with non-silencing siRNA group or control group. To control this reduction was not resulting from a non-silencing siRNA effect ,we compared VEGFsiRNA group with non-silencing siRNA group, showed that the mean tumor volume was reduced by 76.3%. The time for xenografts reached 50mm~3 after injection of the tumor cells in VEGFsiRNA group was delayed by 5.4 days compared with non-silencing siRNA group.The mean doubling time of tumors was not significantly modified by VEGFsiRNA treatments compared with non-silencing siRNA group. The mean doubling time of tumors was delayed by VEGFsiRNA treatments or non-silencing siRNA compared with control group. ELISA of VEGF in tumor contents showed 57.5%reduction in the VEGF expression of tumors from VEGFsiRNA group as compared with non-silencing siRNA group.Conclusion:1 Transient-transfection or stable-transfection of VEGF siRNA plasmid could downregulate the expression of VEGF in human A549 cells in vitro.2 Transient-transfection of VEGFsiRNA-plasmid in vivo with or without PEI or lipofectamine 2000 showed on effect on tumour growth or VEGF in tumour content. Possible reason was low efficiency of transfection in vivo.3 Stable- transfection A549 cell of VEGF siRNA in a xenograft murine modle showed time for xenografts reached 50mm~3 was delayed but tumour growth rate was not inhibited. Part 2 Small interfering RNA (siRNA) targeting VEGF inhibits angiogenesis induced by A549 cell on CAM assaysObjective:To investigate whether blocking the ability of A549 cells to express endogenous VEGF by siRNA could affect VEGF induced angiogenesis in CAM assays.Materials and methods:Fertilized eggs on day 9 were exposed CAM and established CAM model. Chick embryos were randomly divided into four groups(n=12-14): negative control group , positive control group , VEGFsiRNA group , and nonsilencing siRNA group. On day 10, l00μl DMEM or cell culture supernatant were placed on CAM of each group for another 48 hours incubation.. The CAMs were harvested on day12, then fixed and placed on slide under the microscope.Results:The chick chorioallantoic membrane assays have been widely accepted as models for evaluating effects on angiogenesis or anti-angiogenesis.The vascular system of CAM is well developed and directly accessible to observation and experimentation. Compared with negative control group, the proliferation of microvessels was obviously increased when cell culture supernatant with VEGF was added in positive control group , non-silencing siRNA group or VEGFsiRNA group. Positive control group and non-silencing siRNA showed an extremely significant proliferated vessels compared with negative group, as part of the vascular net's morphosis losing the tree type arrangement of vessels. VEGF content of VEGFsiRNA group were only 40%-44% of non-silencing siRNA group or positive control group. Vessels branch points of the chorioallantoic membrane were increased in positive control group by 99% , non-silencing siRNA group by 97% , VEGFsiRNA group by 53% compared with negative group.. Total vessel length of CAM were increased in positive group by55%, non-silencing siRNA group by 49% and VEGFsiRNA group by 45% compared with negative control group.Conclusion:.1. Low concentration of VEGF could induce angiogenensis around the chick chorioallantoic membrane as well as high level VEGF. However ,high VEGF also induced increased bulky and longer of vessel branch and change of vascular network.2. VEGFsiRNA partially inhibited A549 cells induced angiogenesis around the chick chorioallantoic membrane.