Effects Of Targeted Interfere Five Genes On PC12 Cells Proliferation

[Method] We selected three sites of sequences by chemosynthesis, separately targeted for PDXK, VDAC2, RhoGDI-a, TM4,β-sncb (Si-r-PDXK₁,2,3, Si-r-VDAC2₁,2,3, Si-r-RhoGDI-a₁,2,3, Si-r-TM4₁,2,3, Si-r-β-sncb₁,2,3) and one not targeted against any mRNA of the target sites of siRNA as negative control, siRNA SCR (scramble siRNA). We transfected PDXK, VDAC2, RhoGDI-a, TM4,β-sncb siRNA gene into rat adrenal tumor cells (PC 12 cells) using cationic liposomes as transfection reagent Lipofectamine2000TM. RT-PCR were used to select the best interference segment. At 24h after transfection, five kinds of intracellular protein expression were detected by immunohistochemical staining. MTT was used to determine the proliferation, morphology and the numbers of PC12 cell after five gene been interfered.[Results]1. Result of PDXK gene interference:Control with siRNA SCR group, PDXK mRNA expression and cytoplasm PDXK protein were decreased in Si-r-PDXK₁ group, MTT test results indicate that cell viability and cell number were significantly lower in final concentration of 80nM,100nM of Si-r-PDXK₁ groups (P<0.05)2. Result of VDAC2 gene interference:VDAC2 mRNA expression was significantly inhibited in each concentrtrion of Si-r-VDAC2₁,2,3 groups compared with the siRNA SCR group. The results of MTT test indicated that the vitality of cells in the final concentration of 100nM of Si-r-VDAC2 1 and the final concentration of 50nM of Si-r-VDAC2₃ groups were lower than in SCR groups. The cell number was significantly reduced in Si-r-VDAC2₃ 50nM compared with SCR group (P<0.05)3. Result of RhoGDI-a gene interference:Control with the siRNA SCR group, the RhoGDI-a mRNA expression in Si-r-RhoGDI-a₁ 50nM, 100nM groups, Si-r-RhoGDI-a₂ 100nM groups, and Si-r-RhoGDI-a₃ 50nM, 100nM groups were inhibited (P<0.05). Interference of RhoGDI-a gene expression showed no effect on viability and cell number in P12 cell (P>0.05)4. Result of TM4 gene interference:Control with the siRNA SCR group, the TM4 mRNA expression in final concentration of 80nM of Si-r-TM4₂ and the final concentration of 50nM,80nM, 100nM of Si-r-TM4₃ groups were inhibited. MTT test results indicate that cell viability was significantly lower in the final concentration of 50nM of Si-r-TM4₁ groups and final concentration of 80nM and 100nM of Si-r-TM4₃ groups than in SCR group. Cell count showed that the cell number was significantly reduced in final concentration of 50nM,80nM of Si-r-TM4₁ and the final concentration of 50nM,80nM, 100nM of Si-r-TM4₃ control with SCR group (P<0.05)5. Result of 13-sncb gene interference:MTT test results indicate that cell viability was lower in final concentration of 50nM of Si-r-β-sncb 2 and final concentration of 100nM of Si-r-β-sncb₃ groups than in SCR group (P<0.05). The cell number was significantly reduced in final concentration of 80nM of Si-r-β-sncb₃ control with SCR group (P<0.05)[Conclusion]The PDXK, VDAC2, RhoGDI-a, TM4 andβ-sncb gene expression could be decreased by specific siRNA sequences in PC 12 cell, and the growth of PC 12 cell was inhibited, when PDXK, VDAC2, TM4,β-sncb gene expression decreased. Even though RhoGDI-a gene expression was obviously inhibited but that has no effective on growth of PC 12 cell. Our experiment found a vitro RNAi technology, and we detected that four kinds of genes effected the survival and growth of PC 12 cell by this technology, finally these results provided some experimental evidences for researching the function of these genes in vivo after injury.