Mechanisms Underlying Hspd1 Regulates Expression And Alternative Splicing Of Genes Involved In Microglia Activation In Functional Recovery Of Intracerebral Hemorrhage

Objective Intracerebral hemorrhage（ICH）is a subtype of stroke characterized by high rates of mortality and morbidity.The majority of survivors from intracerebral hemorrhage have different degree of function deficit,which results in severe burden to the family and society.To date,the prognosis of intracerebral hemorrhage treated by surgical and medical is unfavorable,especially in function disorder caused by ICH.The mechanism of brain injury after intracerebral hemorrhage is complex,and inflammatory immune response plays a critical role in brain injury post intracerebral hemorrhage.Microglia,as brain resident immune cells,were first to participate in the inflammatory immune response after ICH and play an essential role in injury repair of intracerebral hemorrhage.Therefore,functional status regulation of microglia is crucial for functional recovery post intracerebral hemorrhage.Hspd1 is a RNA binding protein,a member of heat shock proteins,which involved in RNA metabolism and interaction with proteins.The function and molecular mechanisms of Hspd1 involved in intracerebral hemorrhage is unclear.Hence,this study investigate the effect of RNA binding protein Hspd1,regulating differentially expression and alternative splicing of genes in the transcript level,on the activation of microglia and the function recovery of intracerebral hemorrhage.Methods We investigated this project from three aspects.1.Microglia Hspd1 gene knock down analyzed differentially expressed genes and alternative splicing events via high-throughout sequencing data analysis.2.Microglia Hspd1 gene knock down tested the activation and functional status of microglia.3.The role of Hspd1 on functional recovery of intracerebral hemorrhage was explored in clinical specimens and animal experiments.The detailed research methods are as follows:1)Microglia were isolated,cultured and observed the growth morphology of cells by phase contrast microscope.2)Hspd1 sh RNA lentivirus was purchased from Suzhou Gemma Company.Transfected microglia were cultured,RNA and protein were extracted for q RT-PCR and Western blotting test to identify the effect of Hspd1 gene silencing.3)RNA was extracted from microglia for RNA-seq high-throughout sequencing after Hspd1 gene knock down in microglia.Data was processed and analyzed for differentially expressed genes,alternative splicing events and functional enrichment.4)RNA-seq analysis data further verified differentially expressed genes and alternative splicing using q RT-PCR test.5)The activated type of microglia was detected by immunofluorescence,inflammatory cytokine secretion of microglia was obtained by ELASA,M1/M2 m RNA marker of microglia was acquired by PCR,and M1/M2 ratio of microglia was examined by flow cytometry after HSPD1 gene knock down.6)Monocyte cells were collected from peripheral blood of intracerebral hemorrhage patients in 96 hours after cerebral hemorrhage.RNA were extracted and the content of Hspd1 was detected by q RT-PCR.Meanwhile,modified Rankin Scale（m RS）score was evaluated 3 months after ICH,and analyze the correlation between Hspd1 level and neurological function of patients with ICH.7)Stereotaxic apparatus was used to establish intracerebral hemorrhage models,in which adult male SD rats administrated bacterial collagenase IV into caudate putamen nuclei.We evaluated the ICH rat model stability from three different aspects,such as the shape of hematoma of ICH coronal tissue section,MRI examination and behavioral examination of neurological deficit of rats.8)Hspd1 sh RNA lentvirus was injected into the hematoma using stereorientation technique to establish Hspd1 gene knockdown animal models 3 hours after ICH.9)Hspd1 gene knockdown intracerebral hemorrhage animal models were sacrificed 96 hours after ICH.The water content of brain tissue was weighed by wet and dry weight method to evaluate the brain edema,the change of hematoma volume was evaluated by tissue section,and the volume of hematoma was calculated by Image J software.10)Hspd1 gene knockdown animal models of intracerebral hemorrhage were evaluated the neurological deficit and functional recovery at 3,7 and 14 days after intracerebral hemorrhage.Results 1.Microglia Hspd1 gene knockdown by lentivirus was successfully established,and the cell status was fine.The infection efficiency was over 70%,and the infection was successful.2.Microglia Hspd1 gene knocked down by lentivirus,and q RT-PCR results showed that Hspd1 gene knockdown was successful.WB results showed that Hspd1 gene expression decreased significantly in the experimental group in comparison with the control group,and the difference was statistically significant.3.RNA of Hspd1 gene knockdown microglia were extracted for RNA-seq high-throughput sequencing and data processing analysis,resulting in significantly up-regulated expression of 990 genes,which were performed by GO enrichment analysis.Of note,we discovered that Vim alleviates hematoma of cerebral hemorrhage and brain injury,Mfge8 reduce apoptosis and inflammatory response,Lrp1 improves neurological function,Slc2a1 participates in the formation of blood-brain barrier and Atf5 plays a neuroprotective role in noteworthy.The content of 921 genes was significantly down-regulated and was performed by GO enrichment analysis.The pro-inflammatory mediators Hmgb2,chemokine Ccl2,Tardbp and Ezh2 promoting microglia activation and interferon Ifi27l2 a were found,which were worthy of attention.4.5231 significantly different alternative splicing events were found after HSPD1 knockdown in microglia.GO enrichment analysis was carried out on these genes with alternative splicing events.According to the ratio values of gene alternative splicing events were significantly different,and combined with literature,Clk1 involved in microglial neuroinflammation,App involved in cerebrovascular amyloidosis,Gng12,Cast involved in alleviating cerebral hemorrhage and brain injury,and Slc38a1 involved in neuroprotection were found.5.Overlap analysis of differentially expressed genes and differentially spliced genes in this project found that 255 genes were both differentially expressed and differentially spliced,among which 137 genes were up-regulated and 118 genes were down-regulated.GO functional enrichment analysis was performed on these 255 genes.It was found that NF-κB signaling pathway and interferon βsignaling pathway were related to intracerebral hemorrhage inflammation when combined with literature analysis.AS events of enrichment genes in these two pathways were extracted and Cd86,Irgm1 and Mnda1 were finally selected.6.The expression levels of Irgm1,Cd86,Ccl2,Mndal and Ezh2 genes decreased,while the expression levels of Vim and Slc2a1 genes increased in the experimental group compared with the control group,which was significant differences in two-way ANOVA,consistent with expectations.The expression levels of Mfge8 and Lrp1 genes decreased,while the expression levels of Hmgb2 and Tardbp genes increased in the experimental group compared with control group,and there was a significant difference in two-way ANOVA,contrary to expectations.The expression of Atf5 gene did not show significant difference between the experimental group and the control group.7.The probability of alternative splicing events of Irgm1 and Mndal genes was elevated in the experimental group in comparison with control group,and the two-way ANOVA showed significant difference,consistent with the expectation.The probability of alternative splicing events of Clk1 gene in the experimental group was reduced,and there was a significant difference in two-way ANOVA,contrary to expectations.There was no significant difference in the probability of alternative splicing events of Cd86 and Gng12 genes between the experimental group and the control group.8.Hspd1 gene knockdown in microglia were presented with activated state,cell body enlarged,processes shortened,and cell morphology was round or rod-shaped.The expressions of cytokines IL-6 and TNF-ɑ decreased,while the expressions of IL-10 and TGF-β increased in comparison with control group.PCR showed that the expressions of M1 markers CCL3 and i NOS decreased,whereas the expression of M2 maker Arg1 and Ym1 increased.Flow cytometry analysis showed that the proportion of M1 microglia decreased and M2 microglia increased.9.Hspd1 gene expression increased in monocytes of intracerebral hemorrhage,and was negatively correlated with functional recovery of patients 3 months after intracerebral hemorrhage.10.The ICH model was successfully constructed.The hematoma was located in the basal ganglia and its surroundings with clear boundaries and regular morphology,and the limb on the opposite side of cerebral hemorrhage was obviously dysfunctional.11.Hspd1 gene knockdown intracerebral hemorrhage animal model was successfully constructed and Hspd1 expression decreased.12.Hspd1 gene knockdown intracerebral hemorrhage animal model brain edema the amount of hematoma alleviated in comparison with control group.13.Hspd1 gene knockdown can promote neurological function recovery in intracerebral hemorrhage animal models.Conclusions The RNA-binding protein Hspd1 regulates the differential expression and alternative splicing of microglia genes at the transcriptional level,and Hspd1 inhibition promotes the activation of microglia polarization to M2 subtype.The expression level of Hspd1 was negatively correlated with the prognosis of intracerebral hemorrhage.Inhibition of Hspd1 expression could promote the recovery of neurological function in animal models of intracerebral hemorrhage,suggesting that Hspd1 inhibition may play a neuroprotective role in intracerebral hemorrhage by regulating microglia expression.To elucidate the value and potential molecular mechanism of Hspd1 regulation of microglia in the treatment of intracerebral hemorrhage,provide theoretical support and translational level reference for the therapeutic effect of microglia in intracerebral hemorrhage,and provide new possible treatment strategies for the clinical treatment of intracerebral hemorrhage.