The Influence Of SiRNA Targetting CRMP1 On Neurite Growth And Cytoskeleton In Rat Hippocampal Neurons

Objective:To investigate positive effect of CRMP1 gene on neurite outgrowth and cytoskeleton in rat hippocampal neurons.Materials and methods:The three-pair siRNAs acquired via chemical synthesis were screened by RT-PCR and real-time PCR. The effective siRNA interfering CRMP1 gene was supported by immunocytochemical and western blot. The fluorescence radix FAM was constructed upon 5'terminal of the siRNA, and then transiently transfected into hippocampal neurons with lipofection reagent. The localization of FAM and neurite outgrowth of hippocampal neurons were observed by fluorescence microscope. Immunocytochemistry and western blot were used to study the expression of CRMP5 and a-tubulin.Results:(1) Detection of transfection efficiency:The negative control FAM-siRNA was transfected into hippocampal neurons with lipofection reagent and transfection efficiency came up to 52.5% tested by Flow cytometry. (2) Selection of CRMP1-siRNA:Three sequences of siRNA were obtained via chemical synthesis which targeted CRMP1 gene. They could inhibit gene expression of CRMP1 detected by RT-PCR and real-time PCR, compared with the control group. CRMP1-249 siRNA, one of three siRNAs, had the best inhibitory effect on gene expression of CRMP1, and the inhibitory efficiency was 84.3%. Meanwhile, the level of CRMP1 protein had decreased by 22.4%. (3) Distribution of CRMP1-siRNA in cells:After CRMP1-249 siRNA was transfected into hippocampal neurons; green fluorescence was mainly distributed in cell bodies, neurites and growth cones. (4) Results of neurons growth at different time points: After hippocampal neurons were transfected with CRMP1-249 siRNA, cell morphology was observed by fluorescence convert microscope, and the hippocampal neurons emerged obvious morphological changes at 32 h. The cell bodies began to collapse and the neurites were retracted. The length of neurites were shorter than the control group, and the number of them had similar change.There were statistical significance (P<0.05). The hippocampal neurons had dead at 48 h post transfection. (5) Results of confocal laser scanning microscopy and western blot:a-tubulin and CRMP5 proteins were mainly distributed in cell body, neurite and growth cone. And after knockdown of CRMPl with CRMP1-249 siRNA for 24 h, the fluorescence intensity of antigen had been remarkably weakened, as compared with blank control group. This finding was corroborated by western blots, and the levels of a-tubulin and CRMP5 proteins were significantly reduced by 47.7% and 53%. There were obviously different in experimental and blank group (P<0.05).Conclusions:(1) One siRNA Sequence to CRMPl mRNA was screened successfully by RT-PCR and real-time PCR. (2) After inhibition of CRMP1 expression with siRNA, the cell bodies were collapsed and neurites were retracted, leading to cell death. The results suggested that knockdown of CRMPl expression could not promote neurite growth and caused a significant loss of hippocampal neurons. (3) a-tubulin and CRMP5 evenly distributed in the cell bodies, neurites and the growth cones of hippocampal neurons. (4) Inhibition of CRMP1 expression could significantly decrease in the levels of a-tubulin and CRMP5 proteins.