| Objective:Neonatal hypoxic-ischemic brain damage(HIBD)is a major cause of neonatal acute death and chronic nervous system damage.The specific pathogenesis is not completely clear,and there is no specific treatment in clinic.Zinc finger transcription factor ZNF580 is a novel zinc finger protein,and its gene expression level is high in human myocardium,brain tissue,kidney and pancreas.It can mediate cell proliferation,differentiation,migration,survival and apoptosis.Previous studies have shown that ZNF580 expression is increased after hypoxia myocardial injury and inhibits apoptosis of cardiomyocytes,but the role of ZNF580 in neurons has not been reported.The objective of this study was to investigate the expression of zinc finger transcription factor ZFP580/ZNF580 in the in vivo model and the oxygen and glucose deprivation(OGD)model of neonatal rats with hypoxic-ischemic brain injury.The protective effect of lentivirus transfection in oxygen-glucose deprived neurons was investigated.Finally,the possible molecular mechanism of lentivirus in hypoxic-ischemic brain injury neurons was analyzed by RNA-SEQ high-throughput sequencing and bioinformatics,providing a new target for the diagnosis and treatment of neonatal hypoxic-ischemic brain injury.Methods:1.A neonatal rat model of hypoxic-ischemic brain damage was established by RICE method in SD rats at the age of 7 days.Brain tissues were decapitated at different time points after surgery,and part of brain tissues were fixed with 4% paraformaldehyde and embedded in paraffin.Eosinoxylin(HE)staining was used to observe the morphological changes of cerebral tissue on the ischemic side,and TUNEL staining was used to observe the apoptosis of cerebral cortex neurons on the ischemic side,and immunofluorescence staining was used to detect the expression of ZFP580 protein.In the other part,fresh brain tissues were collected and stored at-80 ℃ for RNA and protein extraction,and the expression of ZFP580 m RNA and protein was quantitatively detected by q RT-PCR and Western blot.Behavioral changes were observed in all animals.2.After oxygen and sugar deprivation(OGD),cell activity was significantly decreased(P<0.05).With the extension of OGD time,the activity of SH-SY5 Y cells decreased gradually,and decreased to about 50% of the activity of the control group at 6h.The hypoxia and glucose deficiency time was determined as 6h.The expression of ZNF580 m RNA and protein was significantly increased at 0h,6h,12 h and 24 h after oxygen and glucose deprivation(P<0.05).The level was still high at 12 h and 24 h,and gradually decreased after that.3.Compared with empty vector model group,the cell viability of SH-SY5 Y cells with low ZNF580 expression after OGD treatment was significantly decreased(P<0.01),apoptosis rate was significantly increased(P<0.01),and cleaved caspase-3 protein expression was increased(P<0.01);In contrast,ZNF580 overexpressed cells,after OGD,cell activity was significantly increased(P<0.01),apoptosis rate was significantly decreased(P<0.01),and cleaved caspase-3 protein expression was decreased(P<0.01).4.RNA-seq results showed that 7962 differentially expressed genes were obtained in the ZNF580 overexpressed group and the control group(ZNF580vs CON),of which 3678 were up-regulated and 4284 were down-regulated.A total of 7198 differentially expressed genes were found in the low expression group of ZNF580 compared with the control group(si ZNF580 vs CON),of which 3137 genes were up-regulated and 4061 genes were downregulated.There were 5304 common differential genes between the two comparison combinations,2658 unique differential genes in the ZNF580 vs CON comparison combination,1894 unique differential genes in the si ZNF580 vs CON comparison combination,and 248 in the 5304 co-regulated differential genes.5.Screening and enrichment analysis of differentially expressed genes.Set difference genetic screening conditions for: P value < 0.05 and | log2 Fold Change | > 0.The function and pathway enrichment of differentially expressed genes were analyzed based on Gene Ontology Database and KEGG Pathway Database.6.Thirty-eight differentially expressed genes in the sequencing results were selected for verification with q RT-PCR.Results:1.The successful establishment of neonatal rat hypoxic ischemic brain injury model was verified in terms of behavioral changes,histomorphology(HE staining)and TUNEL staining for cell apoptosis.Compared with Sham group,there were statistically significant differences in Zea Longa scores at 0h,6h,12 h,24h and 48 h in HIBD group(**P < 0.01).HE results showed that the cerebral cortex and hippocampus neurons in the Sham group were orderly and orderly.24 h after HIBD,the neurons in the left cerebral cortex and hippocampus were disordered,with swelling and vacuoloid degeneration.Some cells lost polarity,became round,nuclei deviated,nucleoli and Nissolith were unclear,and some neurons had nuclear pyknosis or disintegration.At 48 h after operation,the above changes were aggravated and the nuclear pyknosis of neurons increased significantly.TUNEL results showed that compared with the Sham group,the apoptosis rate of the HIBD group was significantly increased at 12 h,24h and 48 h postoperatively,with statistically significant differences(**P<0.01,***P<0.001).Compared with Sham group,the m RNA expression of ZFP580 in HIBD group increased gradually at different time points,peaked at 12 h and was still at a high level at 24h(P<0.05),and the protein expression increased at different time points,and was still at a high level at 12 h and 24h(P<0.05).Immunofluorescence staining results showed that compared with Sham group,the fluorescence intensity of hippocampal CA1 region and cortical neurons in HIBD group was significantly increased,indicating that the expression of ZFP580 was significantly increased.2.After oxygen and sugar deprivation(OGD),the cell activity was significantly decreased(P<0.05).With the prolongation of OGD time,SH-SY5 Y cell activity gradually decreased to about 50% at 6h.The m RNA and protein expressions of ZNF580 were significantly increased at 0h,6h,12 h and 24 h after OGD(P<0.05),and were still high at 12 h and 24 h,and then decreased gradually.3.Compared with empty vector model group,SH-SY5 Y cells with low expression of Zn F580 significantly decreased cell activity(P<0.01),increased cell apoptosis rate(P<0.01),and increased cleaved caspase-3 protein expression(P<0.01)after OGD treatment;In contrast,in ZNF580 overexpressed cells,cell activity was significantly increased(P<0.01),apoptosis rate was significantly decreased(P<0.01),and cleaved caspase-3 protein expression was decreased(P<0.01).4.RNA-seq results showed that a total of 7962 differentially expressed genes were obtained in the combination(ZNF580VSCON)between the overexpressed group and the control group,among which 3678 genes were up-regulated and 4284 genes were downregulated.A total of 7198 differentially expressed genes were found in the combination(si ZNF580VSCON)between the low expression group and the control group,among which3137 genes were up-regulated and 4061 genes were down-regulated.There were 5304 common differential genes between the two comparison combinations.There were 2658 unique differential genes in the ZNF580 VSCON comparison combination,and 1894 unique differential genes in the si ZNF580 VSCON comparison combination.Among the 5304 coregulated differential genes,248 genes were in the same direction.5.GO functional enrichment analysis showed that the biological processes involved in248 overlapping differential genes mainly included axon regeneration,regulation of neuronal projection development,regulation of cell morphology,axon development and regeneration,nerve development and regeneration,negative regulation of neuronal apoptosis,cell cycle or cell division,etc.KEGG pathway enrichment analysis mainly involved key signaling pathways including iron death,glutamate synaptic function,endoplasmic reticulum protein processing,estrogen signaling pathway,TGF-β signaling pathway,Npolysaccharide biosynthesis and longevity regulation pathway.6.q RT-PCR verification results showed that in 38 verified genes,and 27 genes were consistent with RNA-seq results,up to 70%,indicating high reliability of RNA-seq results.The results showed that ZNF580 may play an anti-apoptotic role by up-regulating KLF9,IGFBP3 and HSP5 A genes at the transcriptional level.Conclusion:In vivo experiments confirmed the reactive expression of zinc finger transcription factor ZFP580 in neonatal rat model of hypoxic-ischemic brain injury,and the same results were obtained in vitro in human neuroblastoma cell line SH-SY5 Y.Overexpression of ZNF580 and interference of low expression of ZNF580 by lentiviral transfection technology confirmed that ZNF580 can improve the survival rate of oxygen-glycogen deprived cell model and reduce cell apoptosis,so as to reduce neuronal injury.Based on RNA-seq and bioinformatics analysis,it was speculated that ZNF580 may exert its neuroprotective effect by regulating genes and pathways related to neural development,axon guidance,axon regeneration and apoptosis in neurons.Some of the differential genes were verified by q RTPCR,and the results were highly consistent with the RNA-seq results.At the gene molecular level,it was confirmed that ZNF580 may play the role of endogenous protective factor by up-regulating KLF9,IGFBP3,HSP5 A anti-apoptosis genes.It provides an important basis for further research in the future.Innovative: For the first time this research confirmed the zinc finger transcription factor ZFP580 was higher expression in newborn rats with hypoxia ischemia brain damage model;in vitro experiments confirmed ZNF580 could enhance cell viability and reduce the cell apoptosis in OGD cell models;in the genetic and molecular levels,the RNA-seq confirmed ZNF580 can raise KLF9,IGFBP3 and HSP5 A to play the role of the endogenous protective factor.This project was supported by the National Natural Science Foundation of China.The molecular mechanism of micro RNA-206 targeting ZFP580 in regulating phenotypes conversion of smooth muscle cells after balloon injury. |
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