RNAi-mediated gene silencing of vascular endothelial growth factor C suppresses growth and induces apoptosis in mouse breast cancer in vitro and in vivo

Abstract

Vascular endothelial cell growth factor (VEGF)-C promotes tumorigenesis by allowing lymph node metastasis and lymphangiogenesis, among other actions. RNA interference (RNAi) is a novel technique for suppressing target gene expression and may increase the effectiveness of cancer treatments. The present study assessed the influence of VEGF-C RNAi on the apoptosis and proliferation of mouse breast cancer cells in vitro and in vivo. A total of three pairs of small interfering RNA (siRNA) targeting mouse VEGF-C were designed and synthesized prior to transfection into 4T1 cells via a liposomal approach. Reverse transcription polymerase chain reaction, western blot analysis, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining and flow cytometry were performed in vitro to analyze VEGF-C expression, cleaved caspase-3 protein expression and 4T1 cell proliferation and apoptosis. Experiments were also conducted in vivo on BALB/c mice with breast cancer. Tumor weight and volume were measured and the number of apoptotic cells in tumor tissues was assessed by a TUNEL assay. Immunohistochemical assays and an enzyme-linked immunosorbent assay were used to measure the expression of VEGF-C in tumor tissues. The results demonstrated that the three pairs of siRNA, particularly siV2, significantly reduced VEGF-C mRNA and protein levels in 4T1 cells. siV2 was deemed to be the most efficient siRNA and therefore was selected to be used in subsequent experiments. Furthermore, in vitro studies indicated that VEGF-C RNAi significantly decreased cell growth, induced apoptosis and upregulated the expression of cleaved caspase-3 protein. Tumor weight and volume in breast cancer in vivo models was reduced by the intratumoral injection of siV2. Antitumor efficacy was associated with decreased VEGF-C expression and increased induction of apoptosis. The present study therefore indicated that VEGF-C RNAi inhibited mouse breast cancer growth in vitro and in vivo and that it may be a novel targeted therapy for breast cancer.

Proteomic Analysis of Inhibitor of Apoptosis Protein-like Protein-2 on MCF-7 Cell Growth

“…Two groups of mixed small interfering RNA and Lipofectamine 2000 were added to MCF-7 cells in the logarithmic growth phase, and cultivated in 5% CO 2 for 24 hours at 37℃. The small interfering RNA had a nal concentration of 100 nM [13,14]. MCF-7 cells were washed with 0.01M PBS (phosphate buffer saline) to remove the medium.…”

Therapeutic Potency of Nanoformulations of siRNAs and shRNAs in Animal Models of Cancers

“…When the size of tumors reached at ~0.1 cm 3 , the mice were treated with siV 2 (1 µg/gm body weight), scrambled siRNA (SCR) (1 µg/gm body weight) as a negative control and PBS (Blank control) intratumorally for every 2 days. After 3 days of post injection, the mean tumor weights for siV 2 , SCR and controls were 2.272 g, 3.242 g and 3.185 g, respectively, and the tumor volume for either control or SCR was approximately 2.5 cm 3 , whereas that for the siV 2 -treated group was 1.8 cm 3 , thus demonstrating 28% reduction of tumor volume in comparison to the control group [ 120 ].…”

Alternative Splicing in Breast Cancer and the Potential Development of Therapeutic Tools

“…Non-viral carriers [ 40 ] include cationic, anionic or neutral nanoparticles attached to nucleic acids [ 41 ]. Finally, RNA interference-based methods [ 42 ] have successfully silenced genes like CCL2 [ 43 ] and VEGF-C [ 44 ]. It would be interesting to modify some of these methods to target particular splicing isoforms for a more specific outcome.…”