RNA Interference With CRMP5 Inhibites Outgrounth Of Neurite In Rat Hippocampal Neurons

Objective:To select an effective siRNA that interfer with CRMP5 mRNA, and investigate positive effect of CRMP5 gene on neurite outgrowth in rat hippocampal neurons.Materials and methods:After selecting an effective siRNA that interfered CRMP5 gene by PCR, the effective siRNA sequence was acquired via chemical synthesis, and fluorescence radix FAM was constructed upon 5'terminal of the siRNA. The siRNA was then transiently transfected into hippocampal neurons with lipofection reagent. The localization of FAM and neurite outgrowth of hippocampal neurons were observed by fluorescence microscope.Results:(1) The negative control FAM-siRNA was transfected successfully with lipofection reagent and transfection efficiency tested by Flow cytometry came up to 34.3%. (2) Four siRNAs sequence were obtained via chemical synthesis which targeted CRMP5 gene, and two candidate siRNAs were discovered against CRMP5 sequence by RT-PCR technique. By way of Real-time quantitative PCR we obtained an alternative siRNA which silenced the target gene more effective. Confocal microscopy showed that CRMP5 evenly distributed in the cell body, neurite and the growth cone of hippocampal neuron, using labeled secondary antibody exhibited the expression of the CRMP5 in the experimental group was weaker than that in the control group. (3) After the siRNAs which were constructed FAM were transfected into hippocampal neurons distributing in the neuron cell body, neurite and growth cone, hippocampal neurons emerged obvious morphological changes. The length and branches of neurites were shorter than the control group. Meanwhile, compared with the control group, the length and number of neurites and their branches in the experimental group reduced and shorted gradually. Moreover, part cells of the experimental group only had prima-ry neurites. There were statistically significant changes on the length of primary and secondary neurites between the experimental group and the control group at different time points. Conclusions:(1) The candidate siRNAs were transiently transfected into hippocampal neurons of rats with lipofection reagent. (2) An effective siRNA was screened out successfully by PCR technique and it could interfer the mRNA of CRMP5 validly. (3) CRMP5 protein expressed in the cell body, neurite and growth cone of hippocampal neurons. (4) Shorter neurites and less primary and secondary branches in CRMP5-knockdown neurons suggested that RNA interference of CRMP5 could inhibite outgrouth of neurites in rat hippocampal neurons.