| Background and purposeIschemic stroke(IS)is a localized ischemic necrosis of brain tissue due to impaired blood flow to the brain,ischemia and hypoxia.It is the second leading cause of death and disability worldwide and is characterized by a high morbidity,disability and mortality rate.After an ischemic stroke,peripheral circulating leucocytes are recruited and migrate to the ischemic brain tissue to participate in the post-injury inflammatory response.The adhesion of leukocytes to endothelial cells,on the one hand,reduces the flow of red blood cells through the microvasculature,thus causing a "no-reflow" phenomenon after cerebral ischemia and exacerbating brain damage.On the other hand,the recruitment of activated leukocytes leads to neuronal damage through the release of reactive oxygen species,proteases and inflammatory factors.With the development of high-throughput sequencing technology,the role of long non-coding RNAs in ischemic stroke has been gradually revealed,but the role in regulating leukocyte and endothelial cell adhesion after ischemic stroke is not well investigated.Therefore,this research used RNA sequencing to screen for differentially expressed lncRNAs and their target genes in ischemic stroke patients,to investigate the effects of lncRNAs and their target genes on endothelial cell adhesion and inflammatory responses in IS at the population and cellular levels.This will not only provide new targets for the diagnosis and treatment of IS,but also provide a theoretical basis for the etiological study of ischaemic strokes.Methods(1)screening for differentially expressed lncRNAs and their target genes in IS patientsThis study included 53 IS patients and 53 healthy controls from the First People’s Hospital of Zhengzhou City from January 2019 to May 2019,and three of the IS patients and three of the healthy controls were randomly selected to extract total RNA from peripheral blood samples to Guangzhou Ribo Biotechnology Co.for RNA sequencing to identify lncRNA and mRNA expression profiles.Differentially expressed lncRNA and mRNA genes were screened by |log2(Fold change)|>1,P<0.05,and analyzed for GO function and KEGG pathway enrichment.Then,the potential target genes of the differential lncRNAs were predicted using cis-regulation or trans-regulation algorithms and intersected with differential mRNA genes to establish the regulatory relationship between lncRNAs and protein-coding genes.(2)Expanding the population to validate differential lncRNAs and their target genesExpression of differential lncRNA and potential target genes in the remaining 50 IS patients and 50 healthy controls were verified using qPCR to further screen for differential lncRNAs and their target genes.The potential of differentially expressed LINC02649 and its potential target gene ICAM4 as IS biomarkers was analysed by ROC curves.(3)Verification that LINC02649 does not encode a proteinTwo tools,ORF Finder and Coding Potential Assessment Tool,were used to predict LINC02649 codability and confirm LINC02649 as a non-coding RNA.(4)Subcellular localization of LINC02649 in K-562 and THP-1 cellsK-562 and THP-1 cells were subjected to nucleoplasmic separation and the relative expression of LINC02649 in the cytoplasm and nucleus,respectively.The localization of LINC02649 in the cells was verified by in situ hybridization with a fluorescently labeled probe.(5)To investigate the effect of LINC02649 overexpression on the expression of the potential target gene ICAM4 and the adhesion of leukocyte-endothelial cellsA stable LINC02649 overexpression cell line was constructed by lentiviral infection.Detection of cellular overexpression effects and changes in the expression levels of potential target genes ICAM4 by qPCR.The effect of LINC02649 overexpression on the adhesion of K-562 cells to HUVEC cells was examined by fluorescent probe labeling.(6)To investigate the effect of LINC02649 overexpression on the expression of cellular pro-inflammatory and anti-inflammatory factorsThe effect of LINC02649 overexpression on the expression of the pro-inflammatory factors IL-1β,IL-6,IL-8,TNF-α and the anti-inflammatory factors IL-10 and TGF-β1 was examined using qPCR.Results(1)A total of 74 lncRNA genes were differentially expressed between the IS and control groups,of which 23 were up-regulated and 51 were down-regulated in the IS group.A total of 435 mRNAs were significantly differentially expressed between the IS and control groups,of which 139 were up-regulated and 296 were down-regulated in the IS group.The main functions and pathways involved in the screened differential genes were the classical pathway of complement activation,adhesion of homophilic cells by cell membrane adhesion molecules,calcium binding,antigen processing presentation,and PI3K-Akt signaling pathway,which are mainly associated with inflammatory response and cell adhesion.(2)LINC02649 expression was significantly down-regulated in the IS case group(P=0.0011),with an AUC=0.7 for the diagnosis of ischemic stroke;ICAM4gene expression was significantly up-regulated in the IS group(P=0.0002),with an AUC=0.714 for the diagnosis of ischemic stroke.(3)Overexpression of LINC02649 resulted in a significant decrease in the expression of the potential target gene ICAM4(P<0.05)and a significant decrease in the adhesion of K-562 cells to HUVEC cells(P<0.0001).(4)LINC02649 overexpression inhibited the mRNA expression of pro-inflammatory factors IL-1β,IL-6,IL-8 and TNF-α(P<0.05)and promoted the mRNA expression of anti-inflammatory factor TGF-β1(P<0.05).ConclusionLINC02649 overexpression plays a protective role in IS by inhibiting the expression of the cell adhesion factor ICAM4,leukocyte-endothelial cell adhesion and the cellular inflammatory response. |
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