Studies On The Expression And Role Of KHSRP After Sciatic Nerve Injury

ObjectiveTo investigate the expression of KHSRP after sciatic nerve injury, explore the relationship between KHSRP and Schwann cells and neuronal differentiation, analyze the mutual regulation between KHSRP and β-catenin and their roles in Schwann cell and neuronal differentiation, and further to reveal the mechanism of KHSRP underlying peripheral nervous system injury and repair.Methods:1. We made sciatic nerve crush model and used Western blot, immunohistochemistry to study the expression of KHSRP in normal tissue and lesion tissues after sciatic nerve injury in rats; Immunofluorescence staining was used to detect the tissue localization of KHSRP and colocalization with axon specific marker protein NF200 and Schwann cell marker S100, and to analyze the relationship of KHSRP with Schwann cell myelination marker protein Oct-6 after crush and its application related with Schwann cells and neuronal differentiation.2. In order to explore the role of KHSRP during Schwann cell differentiation, we build primary Schwann cell differentiation model in vitro and detected KHSRP, β-catenin and other related protein expression using Western blot and RT-PCR; Nucleocytoplasmic separation found the cytoplasmic transportation of KHSRP; Through the construction of KHSRP small interfering RNA and β-catenin overexpression plasmid and the combination with immunofluorescence, we detected the possiblity of KHSRP in Schwann cells differentiation mediated by regulating the destabilization of β-catenin.3. To further study the action of KHSRP in neuronal differentiation, we constructed neuronal differentiation model by stimulating undifferentiated PC12 cells with 100ng/ml NGF and made the culture of primary DRG neurons to simulate neuronal differentiation. We detected the effect of KHSRP expression in neuronal differentiation, cytoplasmic transportation and neuronal differentiation, maturation.4. In vitro, we made Schwann cells/neuron co-cultures to simulate the process of SCs myelination and detected protein level of KHSRP and β-catenin and contribution of KHSRP cytoplasmic transportation to the stability of β-catenin.Results1. Western blot and Immunohistochemistry showed that KHSRP expression significantly increased after sciatic nerves injury, and reached a maximum at fifth days, then gradually decreased. Immunofluorescence showed that the colocalization of KHSRP with NF200, S100 and Oct-6, which suggested that KHSRP may play a role in neuronal and Schwann cells differentiation.2. During c AMP-induced Schwann cell differentiation, KHSRP expression gradually increased, while the expression of β-catenin decreased. Results of nuclear and cytoplasmic fractions isolation showed that KHSRP had cytoplasmic transfer during the process of differentiation, corresponding with the phenomena of decreased cytoplasmic β-catenin protein level, which hinted that KHSRP may regulate Schwann cell differentiation mediated by action in β-catenin stability. Schwann cells transducted with KHSRP Small interfering RNA had some confused phenogenesis during Schwann cell differentiation and maturation, which morphology was similar with β-catenin overexpressed Schwann cells.3. During differentiation of PC12 cells stimulated by NGF and development of primary cultured DRG neurons in vitro, KHSRP increased gradually at the protein and m RNA expression levels, while β-catenin expression was into an opposite trend. Besides, we also observed cytoplasmic transportation of KHSRP and reduced stability of cytoplasmic β-catenin. PC12 and DRG neurons transducted with KHSRP Small interfering RNA showed defected phenogenesis during the process of differentiation.4. In the procedure of Schwann cells and neurons coculture, KHSRP expression was lowest in 1 day and gradually increased followed with Schwann cell myelination, while β-catenin expression showed a reverse trend. Nucleocytoplasmic fraction separation assay also found nucleocytoplasmic transportation of KHSRP and declined stability of β-catenin.Conclusions1. KHSRP was involved in neuronal and Schwann cell differentiation after sciatic nerve crush.2. KHSRP could effect on the stability of β-catenin by adjusting its own nucleocytoplasmic distribution, all which were associated with neuronal differentiation and Schwann cell myelination process.