A Study On The Effect Of Lentiviral-Mediated SiRNA Silencing NMⅡ Expression In Cultured Rat DRGs Neuros

Objective To construct the NM Ⅱ siRNA lentivirus and explore the effects of NM Ⅱ gene scilencing in cultured rat DRGs neuros.Method Different plasmids of NM Ⅱ siRNA were constructed on the basis of NMⅡ gene sequences, and divide into 5 groups: blank control group, empty vector group,siRNA1 group, siRNA2 group and siRNA3 group. After transfecting into cultured rat DRG neuros, the NMⅡ mRNA expression level was detected by RT-PCR and Western blot was used to detect the expression of NM Ⅱ protein. Meanwhile, the scanning electron microscope technique was used to observe the morphological changes of growth cone.Result We succeeded in designing and building valid siRNA of rat NMⅡ, and packaged lentivirus vectors. Lentivirus vectors carrying the NM Ⅱ siRNAs infected the cultured rat DRG neuros successfully and knock down the NMⅡ expression. The efficiency of NMⅡ gene silencing was more than 80% in siRNA3 group, whereas was about 30% in siRNA1 group and 60% in siRNA2 group. The results of RT-PCR showed that, comparing with other 4 groups, the NMⅡ mRNA expression level in siRNA3 group was downregulated significantly(p<0.05); The results of western blot showed that, comparing with other 4 groups, the NM Ⅱ protein level in siRNA3 group was downregulated significantly(p < 0.05). The results of scanning electron microscope showed that, in the blank control group and empty vector group, the microtubules arranged orderly, intercellular desmosomes were visible clearly, and we can observe the complete shape of growth cones; However, in the siRNA3 group,Most cells dissolved and nerve fibers demyelinated. We failed to find the normal growth cones.Conclusion(1) One siRNA sequence to NMⅡ mRNA was designed and screened successfully,and managed to construct the lentivirus vectors.(2) The NMⅡgene of the cultured rat DRGs neuros could be inhibited by lentivirus mediated siRNA in vitro; NM Ⅱ gene silencing would lead to the cytoskeleton structure changes of growth cones in DRGs neurons and accordinglly caused the collapse of growth cones. Our research indicated that NMⅡ is an effective target in regulating the growth cones cytoskeleton and promoting axon extension.