Screen And Identify The MicroRNA Expression In Repairment Of Corticospinal Tract Injury By Bioinformatics Strategy

Objective: To detect changes in microRNA and target mRNA at the head and tail of the corticospinal tract injury side, and to investigate the function of microRNA in spinal cord injury(SCI)and repairment, which could provide a theoretical basis and experimental evidence in regeneration of the corticospinal tract(CST)after injury.Methods: 1. The left side of the CST in rats was cut off at the level of the pyramid of medullar oblongata. BBB test was used to assess the motor function. 2. After the CST injury, the mRNA expression profile chip and the microRNA expression chip were performed to analyze rostral and caudal gene differential expression in the CSTs respectively. Besides, mRNA and microRNA gene regulatory network of 2 hours and 5 days in damaged rostral and caudal regions were established. 3. Bioinformatics method was conducted to screen differences in microRNA target gene prediction and the intersection of differentially expressed mRNA was selected to conduct the microRNA and mRNA co-expression analysis. Itcould reveal changes at gene expression in the transcriptome level,and it could also surveyed changes at the level of posttranscriptional gene target of microRNA. Gene network database and graph theory were used to construct microRNA and its target genes of potential regulatory relationship networks. 4. Real-time PCR was applied to detect the gene expression of miRNA-124,mi R-342-5p, miR-17-5p, miR-547. 5. Real-time PCR was applied to detect the gene expression of Irf8, Slit1, Ulk1, IL6. 6. Western blot was applied to detect the protein expression of Irf8 and Iba1.Result: 1. Uncoordinated limb movement after CST semitransection, motor function was limited according to BBB test,but not completely paralyzed. There were statistical significances(P<0.05)after comparing 2 hours, 1 day, 3 days, 5 days and 7 days with the control group, indicating the successful establishment of model. 2. According to the microarray results, comparison between2 hours and 5 days in rostral CST, 50 differentially expressed genes were found, including 23 up-regulated genes and 27 down-regulated genes. While comparison between 2 hours and 5 days in caudal region, 57 differentially expressed genes were found, including 20up-regulated genes and 37 down-regulated genes. Abnormalities of microRNA function were obtained at the gene level. 3. Comparison between 2 hours and 5 days in rostral region, we focused on biological processes in regeneration, including immune response,neuronal migration, neural precursor cell proliferation and axonal regeneration. Comparison between 2 hours and 5 days in caudal CST, we also focused on regenerative biological processes such as immune response, induction of apoptosis, glial cell activation and axonal regeneration. 4. Data analysis from qRT-PCR experiments indicated that caudal miRNA-342-5p expression was significantly different(p<0.01) from 2 hours to 5 days, with a reverse correlation with Irf8 expression, whereas no change was observed in rostralmi RNA-342-5p(P>0.05). However, miRNA-124 was significantly different both in rostral and caudal CST(P<0.01). The other two mi RNAs, miR-17-5p and miR-547 verified by qRT-PCR were all statistically different to the control group(P<0.01 or P<0.001). 5.qRT- PCR data analysis showed that the expression of Slit1, Irf8,Ulk1 and IL6 were consistent with the chip results, statistically significant difference compared with control groups(P<0.01 or P<0.001). 6. The results of Western blot indicated that, comparing tissue from 5 days with 2 hours, both Irf8 and Iba1 were significantly increased in caudal samples(P<0.01).Conclusion : After CST injury, there were significant up-regulation or down-regulation differentially expressed genes in rostral and caudal regions. In the caudal region, the results found the mi RNA-342-5p and Irf8 reverse expression, miRNA-17-5p and Ulk1 inversely regulate, miRNA-342-5p and miRNA-17-5p are closely related to SCI repair and regeneration by glial cell activation.While in the rostral region, microarray analysis showed that several chemokines such as IL6 significantly decreased following CST injury, suggesting the possibility of axon regeneration in the rostral region. All findings will provide theoretical base as well as experimental basis for CST injury and repair.