ShRNA Silencing P2X₇ Expression Reduce Metastasis Of P388D1 To Peripheral Lymph Nodes

Objective: Silencing P2X₇ expression by short interfering RNA reduce P2X₇ expression in P388D1 cells. It was analyzed whether P2X₇ is related to the P388D1 metastasis to peripheral lymph nodes.Methods: Analysis of P2X₇ expression by RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR), Flow Cytometry analysis. Next shRNA knockdown of P388D1 cells P2X₇ expression. Experimental metastasis in DBA/2 mice P2X₇ shRNA and control shRNAs were transiently transfected with Effectene transfection reagent following the manufacturer's instructions. Effects of down-regulation on P2X₇ shRNA at 30h after the transfection was assessed with flow cytometry and RT-PCR and immunofluorescence assay. Inbred strain 24 DBA/2 mice, female and male are the same quantity. Being divided into four experiment groups at random, each group has 6 mice. One group is for P2X₇ shRNA, the other is as control shRNA group, the third is blocked P2X₇ by polyclonal anti-P2X₇ Ab. The last group is negative control according to the group of blocked polyclonal anti-P2X₇ Ab by rabbit IgG. Each mouse foot pad injected into cells of 6×10⁵, observed the survival rate and the dissemination condition of tumor cells.Results: RT-PCR, Flow Cytometry analysis and Immunofluorescence assay demonstrated that P2X₇ expression on P388D1 murine lymphoid neoplasm cell line. We measured the level of P2X₇ receptor expression in with transfection shRNA P388D1 cells by Flow Cytometry, RT-PCR and Immunofluorescence. It was shown that expression of P2X₇ by the P388D1 murine lymphoid neoplasm cell line can be silenced by P2X₇ shRNA.Conclusion: Base on our study, it is indicated that P2X₇ has a potential role for dissemination to peripheral lymph nodes. It will open a new vision for the clinic treatment of tumor.