| Objective:To analyze the characteristics of circ RNA-mi RNA-m RNA network in rats with Cerebral Palsy(CP),explore the mechanism of Tuina in treating CP,and look for potential early diagnostic markers of cerebral palsy.Method:Experiment 1: Thirty-eight Sprague Dawley rats born naturally were randomly divided into model group(n=24)and sham group(n=14)on the third day after birth(P3).In the model group,the left common carotid artery was fused and hypoxia was used to establish cerebral palsy model.The second day after modeling(P4),righting reflex was used to verify whether the modeling was successful,and the remaining 17 young rats were randomly divided into two subgroups: cerebral palsy model group(n=8)and Tuina group(n=9).In the sham group,only the skin of the left neck was cut and the common carotid artery was separated,without fusing and hypoxia.The groups were fed in the same environment.On the 61 st day after birth(P61),cerebral palsy model group(n=8)and sham group(n=14)were randomly selected,and white matter(length,width and height ≤0.5cm)was taken from each group.Blood stains and stains were cleaned on the tissue surface,and the liquid on the surface was dried with dust-free tissue paper.Immediately after grouping labeling,they were placed into the pre-cooled and numbered RNase-Free cryogenic cryo-storage tube for high-throughput sequencing.By comparing the data of the cerebral palsy model group and the sham group,the differentially expressed circ RNA in the cerebral palsy model group were screened with P <0.05 as the criterion,and the circ RNA-mi RNA interaction pairs were predicted by bioinformatics analysis,and the mi RNA-m RNA interaction pairs were predicted by the same method,and constructed the circ RNA-mi RNA-m RNA network of cerebral palsy rats.Experiment 2: Select the successful model of the Tuina group of rats in turn by rubbing method,kneading method,twist method on the spine of the rats on both sides of the bladder meridian and limbs,15 minutes each time,once a day,6 days a week,for 8weeks.The operation is done by the same person.On the 61 st day after birth(P61),3 rats were randomly selected from the cerebral palsy model group(n=8)and the Tuina group(n=9)for sequencing,and the remaining rats were collected from white matter and refrigerated for use.Comparing the data of the Tuina group and the cerebral palsy model group,screening the circ RNA and mi RNA up-regulated and down-regulated in the Tuina group compared with the cerebral palsy model group,using bioinformatics analysis tools(Mi Randa,Target Scan),predicting the combination between them and intersecting with the sequencing results,and obtaining the circ RNA-mi RNA relationship pair related to the influence of Tuina on cerebral palsy young mice,further predicting the m RNA that mi RNA may bind,and using cytoscape3.7.2 software to show the cir RNA of the Tuina group.Furthermore,the m RNA data were imported into STRING database and DAVID database,and the genes encoded by m RNA were analyzed by protein interaction analysis,gene ontology function analysis(GO)and signal enrichment pathway analysis(KEGG),so as to analyze the protein molecules,biological functions and signal pathways that may be related to the influence of Tuina on young cerebral palsy mice.Result:1.The circ RNA-mi RNA-m RNA network of cerebral palsy rats contains 29 circ RNAs,of which 9 are up-regulated and 20 are down-regulated;It contains 43 mi RNA,of which21 are up-regulated and 22 are down-regulated;It contains 41 m RNA,including 17up-regulated and 24 down-regulated.2.After 8 weeks of selective spinal Tuina treatment,the nine circ RNAs up-regulated in rats with cerebral palsy all showed a downward trend in the Tuina treatment group,among which cir RNA2294 was significantly down-regulated(P<0.05).There are 8mi RNA bound by cir RNA2294,among which "mi R-103-3p" is up-regulated after Tuina therapy.Circ RNA was down-regulated by 20 in rats with cerebral palsy,and 19 of them were up-regulated in the Tuina treatment group,among which circ RNA1769 was significantly up-regulated(P<0.05).There are 8 mi RNA bound by circ RNA1769,among which the expression of "mi R-129-5p" is down-regulated after Tuina therapy.3.By comparing the two circ RNA-mi RNA-m RNA networks of Tuina and cerebral palsy,it is concluded that the differentially expressed genes in rats with cerebral palsy after tuina treatment are: cir RNA 2294—mi R-30c-2-3p and cir RNA 1769—mi R-129-5p.The gene ontology function analysis(GO)and pathway enrichment analysis(KEGG)of the downstream m RNA target gene showed that there were 194 biological process items,79 cell components items and 65 molecular function items,among which nerve development and brain development were the main gene ontology functions.MAPK signal pathway and c AMP signal pathway are significantly enriched signal pathways.Conclusion:1.The circ RNA-mi RNA-m RNA network of rats with cerebral palsy contains 29 circ RNAs,of which 9 are up-regulated and 20 are down-regulated.It contains 43 mi RNA,of which 21 are up-regulated and 22 are down-regulated;It contains 41 m RNA,including17 up-regulated and 24 down-regulated.2.circ RNA2294 in rats with cerebral palsy was significantly up-regulated and down-regulated after Tuina treatment;Circ RNA1769 was significantly down-regulated in rats with cerebral palsy,and up-regulated after Tuina treatment,and played a regulatory role in mi RNA adsorbed by m RNA target genes.3.Nerve development and brain development are the main biological functions of Tuina affecting the m RNA of rats with cerebral palsy,and MAPK signal pathway and c AMP signal pathway may be the key signal pathways of Tuina affecting rats with cerebral palsy. |
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