Regulation Of MiRNA-138 On SHH Signaling Pathway In Embryonic Spinal Cord Development

Objectives:To investigate the regulation effect of microRNA-138(miRNA-138)on Shh signaling pathway in embryonic spinal cord development in rats,so as to provide experimental basis for exploring potential intervention targets in embryonic spinal cord development.Methods:Bioinformatics analysis:miRDB,miRmap,miRwalk,starBase and other databases were used to predict miRNA-138 target genes,and the intersection of the predicted target genes was online to draw a Wayne map,and functional enrichment analysis(GO)and signal pathway enrichment(KEGG)were performed.In vivo experiments:An experimental model was established by injecting miRNA-138 overexpressed plasmid and miRNA-138 inhibitor plasmid and its control plasmid into the tail vein of pregnant SD rats.Hematoxylin-eosinstaining was used to observe the morphological changes of the embryonic spinal cord.The mRNA transcription and protein expression levels of Shh,Glil and Ptchl in rat embryonic spinal cord were measured by real-time RT-PCR and Western Blot.Results:1.bioinformatics analysis miRDB,miRmap,miRwalk,starBase online database consists of 2 of the same target genes,miRDB,miRmap,miRwalk online data a total of 21 of the same target genes,then on the 21 of the same target genes KEGG,GO analysis found that the miRNA-138 main enrichment in the transcription regulation of gene function,RNA metabolism,gene expression and biological macromolecular synthesis;The signaling pathways involved in KEGG are mainly cancer pathway analysis,axonal guidance,chronic myelogenous leukemia,neurotrophic factor signaling pathway,autophagy,Notch and Wnt signaling pathway.2.HE staining:After the overexpression of miRNA-138,the nucleoli of fetal spinal cord nerve cells were more closely arranged,the cell body area increased,and some neuron nuclei appeared nuclear shrinkage phenomenon and neurofibrillary distortion after the inhibition of miRNA-138.3.Immunofluorescence:Shh immunoreactive positive cells around the central canal of spinal cord in fetal rats showed relatively strong activity.The number of Shh immunoreactive positive cells increased after the overexpression of miRNA-138,and the decrease of Shh immunoreactive positive cells after the suppression of miRNA-138 was statistically significant compared with the control group(p<0.01).4.real-time fluorescent quantitative PCR:gestational age of 17 d,19 d fetal sd rats spinal cord tissues express group increased transcription of the miRNA-138,the peak of 17 day after increases with time gradually decline,compared with the control group with statistical difference(p<0.05),spinal cord tissue inhibitor group the miRNA-138 transcription in gestational age 17d began to decline,after increases with time gradually rise,compared with the control group was statistically difference(p<0.05);The mRNA level of Shh in the spinal cord of fetal SD rats with the gestational age of 17d and 19d after the overexpression of miRNA-138 showed a statistically significant difference compared with the control group(p<0.01),while the inhibition of miRNA-138 showed an opposite result.The mRNA level of Glil and Ptchl mRNA in the Shh signaling pathway showed no change and no statistical difference compared with the control group(p>0.05).5.Western Blot:After the overexpression of miRNA-138,the expression of Shh protein in the spinal cord of fetal rats was increased at 15d,17d and 19d,which began to decdifference(p<0.05),and rease after 19d,and then gradually returned to normal.Compared with the control group,there was a statistical the effect of inhibiting miRNA-138 was opposite.In Shh signaling pathway,Glil and Ptch1 protein expressions at 15,17,19 and 21 days of gestational age did not change,and there was no statistical difference compared with the control group(p>0.05).Conclusions:There is a positive correlation between miRNA-138 and Shh in the development of rat embryonic spinal cord.The regulation of miRNA-138 on Shh may be one of its mechanisms to promote the development of embryonic spinal cord.