Study On The Expression Of Folate Receptor In Tumor Cells And Specific Delivery Of SiRNA To Tumor Cells

ObjectiveFolate receptors serve as target marker of tumor cells. Therefore, we detected the expression of folate receptor in tumor cells. The reporter genes, luciferase and GFP were used to study the effects of siRNA via folate receptor. Methods1. Folate-pRNA labeled FITC was used to detect the expression of folate receptor in tumor cells by flow cytometry and inverted fluorescence microscope.2. As a control for nonspecific inhibition, pRNA/siRNA(GFP)was used. The interfering effects of pRNA/siRNA(firefly luciferase) were testified by cotransfection with pGL3-control, pRL-TK and pRNA/siRNA(firefly luciferase) using Lipofectamine 2000.3. As a control for nonspecific inhibition, pRNA/siRNA(luciferase) was used. The interfering effects of pRNA/siRNA(GFP) were testified by cotransfection with pEGFP-N2 and pRNA/siRNA(GFP) using Lipofectamine 2000.4. Folate-chimeric dimer complex containing folate-pRNA and pRNA /siRNA(firefly) was incubated with 01 tumor cells. Luciferase activities were measured by dual-luciferase reporter assay system.Results1. The results obtained from flow cytometry revealed that 01 and 02 tumor cells overexpressed folate receptors among 25 kinds of cell lines detected.2. pRNA/siRNA(luciferase) were cotransfected into 01 cells with both plasmid DNA pGL3 encoding firefly luciferase and pRL-TK encoding Renilla luciferase. The exprimental results revealed that pRNA/siRNA(luciferase) suppressed its target gene efficiently and specifically. The expression level of firefly luciferase declined apparently. No silencing of the luciferase genes occurred when pRNA/siRNA(GFP)were introduced into cells. Another group was that pRNA/siRNA(GFP)was cotransfected with pEGFP-N2. pRNA/siRNA(GFP)effectively inhibited GFP gene expression. In contrast, such inhibitory effects were not observed with a control construct, pRNA/siRNA(luciferase). The data showed that the effects of siRNA were specific.3. The nanoparticles fabricated by folate-pRNA and pRNA/siRNA(firefly luciferase) can enter into the cells directly. Then siRNA moiety can also specifically induce the interfering effects .ConclusionspRNA/siRNA were introduced into tumor cells, using Lipofectamine 2000. The results revealed that pRNA/siRNA effectively inhibited target gene expression in a dose-dependent manner. Folate receptors were overexpressed in certain tumor cells and served as tumor specific marker. The folate molecule was formed a dimer with a pRNA/siRNA chimera to achieve specific delivery. pRNA/siRNA targeting reporter genes can specifically induce the effects of RNAi. The pRNA chimera will be promising reagents in tumor biological therapy.