Effect Of VEGF-C Silencing On MCF-7Cells Growth In Vitro And In Vivo

Part1. Construction of Lentivirus-Mediated Small Interfering RNA Vectors and observed the VEGF-C expression on MCF-7cellsObjective:The aim of this study is to construct lentivirus vector containing GFP reporter gene drived by VEGF-C gene and to transfect different kinds of cells, then observed the ransfection efficiency. To study the effect of knockdown VEGF-C expression on MCF-7cells interfered with Lentivirus-Mediated Small Interfering RNA Vectors.Methods:Lentiviruses were produced by293T cells following the instruction. Virus supernate was collected and concentrated, then tested the titer of virus by hole by hole dilution method. Used the lentivirus vector to transfect different kinds of cells, then observed the ransfection efficiency. Lentivirus-Mediated Small Interfering RNA Vectors were transfected into breast cancer MCF-7cells, Real-time PCR was performed to detected the mRNA expression of VEGF-C, the proportion of trasfected cells and the knockdown rate of VEGF-C were calculated.Results:(1) The transfection efficiency is extremely high with Lentivirus-Mediated Small Interfering RNA Vectors for many kinds of cells, and the transfection efficiency of MCF-7cells was more than80%;(2) The mRNA expression of VEGF-C was decreased by nearly50%.Conclusions:The transfection efficiency is extremely high with Lentivirus- Mediated Small Interfering RNA Vectors, and VEGF-C gene expression was knocked down efficiently. Part2. The effect of VEGF-C/SiRNA on cell proliferation and apoptosisObjective:To study the. effect on cell proliferation and apoptosis after knockdown VEGF-C expression on MCF-7cells interfered with Lentivirus-Mediated Small Interfering RNA Vectors.Methods:The MCF-7cells were divided into3groups:the SiRNA transfected group, nonsense siRNA transfected group and the normal MCF-7cells group. Proliferation of cell was measured by MTT assay. The apoptosis and Cell cycle was analyzed by FCM. The data were analyzed by one-way ANOVA of SPSS13.0for windows.Results:(1) The proliferation of the VEGF-C/SiRNA cells was inhibited;(2) Cell cycle analysis showed less cells in S phase (13.47%±1.88%) and more cells in G0/G1phase (70.92%±2.42%) than control groups(P<0.05);(3) The apoptosis rate (29.23%±3.14%) of the VEGF-C/SiRNA MCF-7cells was increased significantly.Conclusions:After the transfection with Lentivirus-Mediated Small Interfering RNA Vectors, cells proliferation was inhibited and cells apoptosis was increased. Part3. Suppress the tumor formation of MCF-7cells in nude mice xenografts by silencing of VEGF-CObjective:To investigate the effects after knockdown the VEGF-C expression on MCF-7cells interfered with Lentivirus-Mediated Small Interfering RNA Vectors in nude mice models.Methods:Thirty nude mices were received subcutaneously injection of0.1ml(1.0×107/ml) MCF-7cells which transfected with SiRNA Vectors、 negative control vectors and nothing respectively. The tumor sizes were measured each5days and calculated by the formula:volume(mm3)=1/2length×(width)2. After a35-day follow-up period, the mice were exterminated, and then weight the tumor volumes. The expression of VEGF-C、MVD and LVD in tumors grow in nude mice was confirmed by immunohistochemistry. The data were analyzed by one-way ANOVA of SPSS13.0for windows.Results:(1) Tumor formation rate and weight of tumor volume in knock down group were a little lower than that of other groups, but there are not statistically significant difference(P>0.05); The tumor growth curve showed that there is not much difference from the three groups;(2) The VEGF-C gene expression in knock down group was reduced significantly;(3) Microvessel density in knock down group was (8.83±1.72),were (11.17±2.48) and (11.02±1.41)in negative control group and blank control group (P>0.05).(4) Lymphatic vessel density in knock down group was (5.33±1.63),were (8.17±1.83)and (8.50±1.05) in negative control group and blank control group (P<0.05)Conclusion:The transfection of siRNA silencing VEGF-C expression on MCF-7may be could inhibit tumor growth by reducing lymphangiogenesis in nude mice models. However the silencing of VEGF-C gene had no effect on subcutaneous tumor angiogenesis.