Facilitation of dendritic cell differentiation using RNAi system to down-regulate VEGF secretion of tumor cells Free

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Purpose and introduction: Multiple studies have suggested that the “immunosuppressive” cytokines secreted by tumor cells play significant roles in tumor-induced immunosuppression. Vascular endothelial growth factor (VEGF), which induces angiogenesis, has been shown to be one of the tumor-derived immuno-suppressive factors inhibiting the differentiation of dendritic cells (DCs). DCs are the most potent antigen-presenting cells and appear to play a central role in anti-tumor immunity. Therefore the inhibition of differentiation from DC precursurs to DC by VEGF is considered to play some roles in the suppression of the immune responses against tumor cells. The RNA interference (RNAi) is a sequence-specific posttranscriptional gene silencing mechanism, which is triggered by double-stranded RNA (dsRNA) and causes degradation of mRNA homologous in sequence to the dsRNA. Recently, it was reported that expression vectors using U6 or H1 promoter carring the small interfering RNA (siRNA), which consist of 192̃3b.p dsRNA could down-regulate target gene in sequence specific manner in mammalian cells. In this study, we examined whether the inhibition on the DC differentiation could be prevented with the inhibition of VEGF using RNAi system. Experimental Design and Results: The siRNA expression vectors carrying five different siRNA sequences targeting murine VEGF (mVEGF) were constructed. When these siRNA expression vectors (pcU6imVEGF-Seq1 –Seq5) were transfected on MCA205 with Effectene transfection reagent, the secretion of mVEGF on MCA205 was down regulated with pcU6imVEGF-Seq2 and -Seq5. To examine DC-tumor interaction, MCA205 and DC precursors were co-cultivated using trans-well plates for 4 days. Although DC differentiation, represented by CD11C expression, was inhibited in the co-culture with MCA205 transfected with mock vectors (pcU6icassette) and negative control vectors (pcU6iGLB3), this inhibition was prevented when DCs were co-cultured with MCA205 transfected with pcU6imVEGF-Seq2. Conclusions: Using co-culture systems consisting of DC precursors and MCA205, the DC differentiation could be facilitated, when the secretion of mVEGF on MCA 205 cells were down regulated with pcU6imVEGF-Seq2. These results indicated that RNAi systems could be used for the regulation of immune responses affected by tumor cells.