Studies On HIE Differentially Expressed Genes And Their Mechanisms Based On Transcriptome Sequencing

Hypoxic-ischemic encephalopathy(HIE),a brain injury caused by perinatal asphyxia is one of the most common diseases leading to dysfunction of the central nervous system of newborn.The pathological and physiological changes of hypoxic ischemic brain injury include energy metabolic failure of brain cells,autonomic regulation of cerebral vessels,excitatory amino acid neurotoxicity,increased production of oxygen free radicals,increased release of inflammatory cytokines,and activation of apoptotic genes.These elements link closely,interact as cause and effect,overlap into each other,and form a vicious cycle that eventually leads to the death of nerve.Currently,the main methods for treatment are supportive therapy,control of convulsion and treatment of cerebral edema.But no effective and specific drug is available.Therefore,advanced understanding into the molecular mechanism of the sickness is needed,as well as identifying new targets and methods for the treatment.Objectives:The purpose of this study was to investigate the differential expression of total transcriptome in the occurrence of hypoxic ischemic encephalopathy,and to construct the differential gene expression profile.Bioinformatics methods were used to screen and verify HIE regulatory targets and further explore the mechanism of Nrf2 regulation on brain protection.Methods:In this study,7-day-old newborn rats were studied.The classic modeling method used was:The left common carotid artery was permanently ligated after anesthesia(double ligation or snipping of the common carotid artery after double ligation)in a 7-day-old newborn rat.After 2 hours post operation recovery,the rat was placed in an anoxia box with 8%oxygen concentration for 2 hours.Preoperative prophylactic administration drug(TSA)was applied 3 days in advance.The brain tissue sample was taken 24 hours after the operation.Full transcriptome sequencing was used to construct the differential gene expression profile.The data were sequencing by using biological clustering,sequence alignment,path analysis and other methods.The sequenced data were verified by RT-PCR.At the level of general animals,identification of the morphological changes of neurons was done by HE staining,neuronal Nissl staining and TUNEL detection.The activity of SOD and MDA was detected by ELISA to assess oxidative stress injury.Western Blot and RT-PCR were used to detect the regulatory effect of TSA on important genes in Nrf2/HO-1 anti-oxidative stress pathways and NLRP3 inflammasome.In vitro,the OGD/R cell modeling was performed using primary hippocampal neurons.MTT,LDH,Caspase3,SOD,MDA and ROS were detected to determine hypoxic ischemic injury.Luciferase reporter gene assay was used to confirm the targeting effect of miR-153 on Nrf2.RNAi technology was used to interfere with Nrf2 expression in neuronal cells.After anti-miR-153 treatment,cellular oxidative stress damage was detected to clear the relationship of upstream and downstream effects of Nrf-2 and miR-153Results:1.Transcriptome sequencing and bioinformatics analysis were applied to screen out a total of 1067 significantly different genes,among which 935 were up-regulated and 132 were down-regulated.2.DEGs GO analysis:Significant biological-processes in which differentially expressed genes are mainly concentrated include inflammatory response,infection and immune response.Differences in gene enrichment of Top 15 GO include:inflammatory response,defense response to virus comes,innate immune response and cellular response to lipopolysaccharide,NLRP3 inflammasomes complex,etc.NLRP3 gene was involved in all the above pathways.KEGG enrichment analysis showed that there were significant differences in the nod-like receptor signaling pathway.3.mRNA levels of differentially expressed genes in brain tissue of hypoxic ischemic newborn rats were detected by RT-PCR.Compared with the control group,the expression genes related to inflammatory complex and oxidative stress changes significant.The mRNA levels of NLRP3,ASC,Caspase1,,1L1β and IL18 were significantly up-regulated,and Nrf2,and HO-1 were significantly down-regulated.4.We established the HIE model of newborn rats,used TSA to regulate their histone acetylation level,and explored the mechanism of histone acetylation to regulate Nrf2 in the HIE of newborn rats.The changes of inflammasome,TXNIP and Nrf2 signal pathway were detected by Western Blot and RT-PCR.The results showed that the protein expression level of Nrf2 was decreased,and the gene expression levels of Nrf2 and HO-1 were lower than those of the control group.The protein and gene expression level of TXNIP were decreased.The protein levels of both NLRP3 and RIP3 were increased in HIE group,and the gene expression levels of NLRP3,RIP3,ASC,Caspasel,IL1β and IL18 were also increased.After TSA treatment,compared to HIE,Nrf2 protein and gene expression level were up-regulated.Protein levels of both NLRP3 and RIP3 were decreased,and gene expression levels of NLRP3,RIP3,ASC,Caspasel,IL1β and IL18 were also decreased.From the above results,histone acetylation regulating Nrf2 in newborn rats HIE plays a protective role.5.We established a neuronal OGD/R model and studied the regulation of miRNA to Nrf2 by using miR-153 as the entry point.Western Blot and RT-PCR were used to detect the changes of miR-153 gene level and Nrf2 signal pathway.The results showed that comparing to the Control group,the expression level of miR-153 in OGD/R group was increased in 12 hours and significantly increased in 24 hours,while the expression level of miR-153 in neurons was significantly decreased after anti-miR-153 treatment.And compared to the Control group,the survival rate of neurons decreased after OGD/R induction,LDH,Caspase3,ROS,and MDA levels increased,but SOD decreased.After anti-miR-153 treatment,cell viability increased,LDH,Caspase3,ROS,MDA levels decreased compared with the control group,and SOD activity increased.After inhibition of miR-153,Nrf2 protein,mRNA expression were up-regulated,and its downstream molecules including ARE activity and HO-1 gene expression were simultaneously up-regulated.The expression of Nrf2 and HO-1 were inhibited after the simultaneous transfection of Nrf2 siRNA with the inhibition of miR-153,and intracellular ROS and Caspase3 were significantly increased.It indicated that miR-153 played its role through the Nrf2 signaling pathway.Based on the above results,miR-153 can regulate the Nrf2 signal pathway in the OGD/R of neurons to play an important role.Conclusion:1.HIE in neonatal rats is accompanied by changes in gene expression of biological processes such as inflammatory response,infection and immune response.2.In neonatal rat HIE,the expression of NLRP3 inflammasome-related genes was significantly changed,and NLRP3 inflammasome may be involved in HIE biological processes.3.In neonatal rat HIE,Nrf2/HO-1 participating in HIE can be a potential therapeutic target.4.Histone acetylation regulates the expression of Nrf2 and NLRP3 inflammasome in the brain of HIE rats and reduces neuronal damage.5.In OGD/R-induced neuronal injury model,anti-miR-153 exerts a protective effect on neurons through Nrf2.