Research Of MiR-301a-3p In Pathogenesis Of Bacterial Meningitis By Targeting To Cx43 Gene

Bacterial meningitis(BM),as a serious infectious disease of the central nervous system,remains a major cause of morbidity and mortality,which is common in infants and young children.With the lack of resistance in blood-brain barrier,the bacteria enters the brain nervous system much easily.75% bacterial meningitis were found in children younger than 5 years old and it was estimated by World Health Organization(WHO)that about 170 thousand children die from bacterial meningitis every year worldwide.Therefore,to explore the pathological mechanism of bacterial meningitis and to find new therapy of bacterial meningitis is of great significance.Gap junction is the transmembrane channels formed between two adjacent cells,the gap junctional intercellular communication would regulate the exchange of substances and signals between cells,concerned with cell growth,differentiation,immune response,as well as many physiological and pathological processes.There were more than twenty kinds of proteins that have been found in mammals,among which Connexin 43(Cx43)is the most abundant and widely distributed in astrocytes and plays an important role in the material exchange and electrical signals transmission of cells.It is found that abnormal expression of Cx43 is related to many pathophysiological abnormalities such as skin burn,cardiovascular disease,tumor,reproductive system diseases,which was also related with epilepsy,brain tumors,brain injury,depression,brain glioma and other neurological diseases.Astrocytes arethe majority cells of the central nervous system.It has been found that Cx43 expressed abnormally in astrocyte mediated inflammatory response,thus we hypothesized that Cx43 also plays a role in the bacterial meningitis.MicroRNAs(miRNAs)are endogenously expressed noncoding RNAs in eucaryote cells,the pair-bond of target gene 3’UTR region could regulate the gene transcription,with important biological and pathological functions and it is found that more than 30% of protein may be regulated by miRNA.We’ve found that Cx43 may be the target gene of miR-301a-3p by bioinformatic method(TargetScan and MiRanda target gene prediction database).We will study the expression of miR-301a-3p and Cx43 in bacterial meningitis and find the effect of miR-301a-3p on Cx43 regulation.This study includes the following four parts: the first part is expression of miR-301a-3p and Cx43 in bacterial meningitis model of rats;the second part is biological effects of miR-301a-3p in astrocytes;the third part is preliminary study of post-transcriptional mechanism of miR-301a-3p regulating Cx43;The fourth part is the effects of miR-301a-3p in bacterial meningitis model.The first part: expression of miR-301a-3p and Cx43 in Bacterial meningitis model of rats Methods:1.Bacterial meningitis model of rats was prepared,60 rats were randomly divided into control group(n=20),24 h model group(n=20),48 h model group(n=20),5d model group(n=20).Compared crainal spinal fluid leukocyte counts of each group,the mortality and Loeffler neurobehavioral score were also recorded.2.ELISA method was used to detect expressions of IL-6,IL-8,TNF-α in the brain of different groups.3.Immunohistochemical and western blot method were used to detect expressions of Cx43 protein,Using the qRT-PCR to detect expressions of Cx43 mRNA.4.qRT-PCR method was used to detect expressions of miR-301a-3p.Analyzethe correlation between expression level of miR-301a-3p and Cx43.5.Using SPSS 21.0 software to process statistical analysis with inspection levelа= 0.05.Results:1.The Loeffler score decreased with time in bacterial meningitis group,which was also lower in bacterial meningitis group than control group at the same time point(P<0.05).2.The results of ELISA showed that IL-6,IL-8 and TNF-α were higher in bacterial meningitis group than the control group at 24 h,48h and 5d with significant difference(P<0.05,).The expression of IL-6,IL-8 and TNF-α gradually increased with the time in bacterial meningitis group.3.Immunohistochemical results showed that the Cx43 protein in the control group was weak positive,After modeling,more cells expressed Cx43.The results of Western blot showed that the expression of Cx43 was higher in bacterial meningitis group than control group(P<0.05)and it reached the peak at 24 h after modeling,then it decreased with time.4.The relative expression of miR-301a-3p was higher in bacterial meningitis group than control group in RT-PCR(P<0.05),which increased with time and showed significant difference at different time point(P<0.05).The second part: biological effects of miR-301a-3p in astrocyte Methods:1.MiR-301a-3p agomir,miR-301a-3p antagomir and miR-NC were synthetized and transfected into astrocytes.2.Detect proliferation and growth ability of cells in each group using CCK8.Use AnncxinV-FITC/PI double marker flow cytometry,Caspase 3 to test apoptosis of cells in each group.3.Using western blot method to detect the expression of Cx43 protein.Results:1.The cells transfected by miR-301a-3p were with greater proliferative capacity than Control as well as miR-NC group(P<0.05)while the cells transfected by miR-301a-3p antagomir were with less proliferative capacity than the other 2groups(P<0.05).2.The results of flow cytometry showed that apoptosis of the cells that transfected by miR-301a-3p agomirs was inhibited while the cells that transfected by miR-301a-3p antagomir was accelerated(P<0.05).3.Western blot showed that the Cx43 expression was inhibited by miR-301a-3p agomir transfection.The third part: preliminary study of post-transcriptional mechanism of miR-301a-3p regulating Cx43Methods:1.Predict the potential target gene of miR-301a-3p by bioinformatics analysis.2.Construct the recombination vector wt-pEZX-MT05-Cx43,mut-pEZXMT05-Cx43.Use dual report experiment to validate the target genes of miR-301a-3p.3.Construct the expressional vector pcDNA3.1-Cx43 and analyzed the biological activity regulation of miR-301a-3p on astrocytes by restoring experiment.Results:1.Verify Cx43 is the target gene of miR-301a-3p by bioinformatics analysis.2.Dual report vector experiments show that miR-301a-3p can bond with 3’UTR regions of Cx43 and regulate its expression negatively.3.Transfected the recombination vector pcDNA3.1-Cx43 without 3 ’UTR regions of Cx43 into astrocytes led to the restoration of the negative function ofmiR-301a-3p on Cx43 and led to apoptosis of astrocytes.The fourth part: the effects of miR-301a-3p in bacterial meningitis model of rats Methods:1.Bacterial meningitis model was prepared,The rats were randomly divided into model group(n=10)and miRNA group(n=10),The miRNA group was treated with miR-301a-3p agomir,10 normal rats were used as Control.2.The Loeffler score of each group were compared.Using the ELISA method to detect expressions of IL-6,IL-8,TNF-α in different groups.Using the western blot method to detect expressions of Cx43 protein.Results:1.The Loeffler scores of the model group and the miRNA group were lower than the control group,while Loeffler scores of the miRNA group were higher than the model group(P<0.05).2.The results of ELISA showed that IL-6,IL-8 and TNF-α were higher in the model group and the miRNA group than the control group with significant difference(P<0.05)while the miRNA group was lower than the model group.3.The results of Western blot showed that Cx43 protein expression were higher in the model group and the miRNA group than the control group with significant difference(P<0.05)while the miRNA group were lower than the model group.Conclusion:1.The expression of miR-301a-3p and Cx43 in bacterial meningitis brain of rats both increased,while it showed negative correlation with them.2.We transfected miR-301a-3p agomir into astrocytes and found that the proliferation of cells was increased and the ability of apoptosis was inhibited.Therefore,miR-301a-3p may play an important regulatory role in the development ofbacterial meningitis.3.miR-301a-3p binds to the 3’UTR of Cx43 mRNA and regulates its expression,thus playing a corresponding role in bacterial meningitis.4.miR-301a-3p is expected to become a new target for bacterial meningitis therapy.