Analysis Of MiRNA-mRNA Expression In The Hippocampus Of Alcohol-dependent Rats And Verification Of MiR-96-5p Target Gene And Function

BackgroundIn recent years,research on alcohol-dependent miRNA regulation has gradually become a hot spot.Most genes are regulated by miRNAs,and miRNA is abundantly expressed in brain tissue.They participate in processes such as neuron proliferation,differentiation,synapse formation,plasticity and affect cell adaptability caused by substance dependence.The study found that excessive alcohol intake induces abnormal miRNA expression in rats.miRNA regulates the formation of alcohol dependence by regulating a variety of neurotransmitter-related proteins and nutritional factors.miRNA plays an important regulatory role in the evolution of clinical or behavioral characteristics(craving,withdrawal,re-drinking,cognitive impairment,etc.).However,the role of miRNA and target genes in the formation of alcohol dependence and in the evolution of clinical characterization needs to be further clarified.ObjectivesScreening of differentially expressed miRNA and mRNA in the hippocampus brain region of alcohol-dependent model rats.Validate the regulatory effect of miRNA on target genes.Explore the role of miR-96-5p in regulating Tp73 and further study the effect of miR-96-5p on the apoptosis of nerve cells.Methods1.Total RNA was extracted from hippocampal brain area of rats in alcohol-dependent group(n=6)and control group(n=6),and miRNA and mRNA were sequenced byhigh-throughput sequencing method to screen out differentially expressed genes.Analyze the correlation between miRNA and mRNA to build a miRNA-mRNA negative regulatory network.2.Use NCBI,GCBI and other databases to screen for differentially expressed mRNA in the hippocampus brain region of alcohol-dependent model rats.Using the miRNA-mRNA negative regulatory network and software miRWalk to predict miRNA,and select miRNA-mRNA.3.Using quantitative Real-time PCR technology to verify and screen mRNA and miRNA expression at the tissue level.4.Treated PC12 cells with miRNA mimic or inhibitor,the targeted regulation of miRNA on mRNA is verified by quantitative Real-time PCR experiments at the cellular level.5.Western Blot assay was used to detect the protein level of Tp73 after miR-96-5p intervention,and dual-luciferase reporter gene assay was us ed to detect the binding of miR-96-5p to the 3 'untranslated region of Tp73,to verify the miR-96-5p targeted regulation of Tp73.6.With different transfection concentrations and different transfection times,CCK-8kit was used to detect the change of cell survival rate after miR-96-5p inhibitor transfection.7.Flow cytometry was used to detect the effect of miR-96-5p inhibitor transfection on PC12 cell apoptosis.Results1.The results of high-throughput sequencing in the hippocampal brain region of alcohol-dependent rats: 47 mRNA were significantly differentially expressed,of which 16 genes were significantly up-regulated and 31 genes significantly down-regulated;38miRNAs were significantly differentially expressed,of which 4 miRNA were up-regulatedand 34 miRNA down-regulated.The correlation between mRNA and miRNA differential expression was further analyzed to construct a miRNA-mRNA negative regulatory net-expression(P<0.05).2.Using the database to screen out genes may be associated with alcohol dependence,substance addiction,neuro-development and nerve cell apoptosis,such as Clic6,Tp73,Pla2g3,Ak7,Gli1,Cckbr,miR-6215,miR-183-5p,miR-96-5p,miR-196a-5p,miR-206-3p and miR-34b-3p.In the hippocampus of alcohol-dependent rat models,the screened genes expression were verified by quantitative Real-time PCR.The results showed that the sequencing datas were basically consistent with the laboratory verification(P<0.05).3.Initially verifying the targeting effect of miRNA on mRNA at the cell level: the expression levels of miRNAs in the hippocampus brain region of alcohol-dependent model rats were simulated by miRNA mimic/inhibitor to interfere with cells,up-regulate/down-regulate miRNAs.quantitative Real-time PCR results show that miR-96-5p inhibitor target gene Tp73 expression is up-regulated,miR-34b-3p mimic target gene Cckbr expression is down-regulated,which finally confirmed the targeted regulation of the two groups of miRNA-mRNA(P<0.05).4.After transfection of miR-96-5p inhibitor,the protein level of Tp73 was significantly increased comparing to the control group by Western blot.Using the dual-luciferase reporter gene assay,the reported fluorescence of the wild-type ve ctor r-Tp73-WT was significantly down-regulated after transfection with miR-96-5p mimic comparing to the control group;mutations were made to its predicted target site,and the reported fluorescence in the mutant vector r-Tp73-MUT showed a clear return(P<0.05).5.The survival rate of cells transfected with miR-96-5p inhibitor at different transfection concentrations and different transfection times were less than that of the control group through the CCK-8 experimental result.Compared with the control group,the number of apoptosis of PC12 cells in the miR-96-5p inhibitor transfection group increased(P<0.05).Conclusions1.miR-6215,miR-183-5p,miR-96-5p,miR-196a-5p,miR-206-3p,miR-34b-3p,Clic6,Tp73,Pla2g3,Ak7,Gli1,and Cckbr are differentially expressed in the hippocampal brain region of the alcohol-dependent rats model.2.Preliminary verification of miR-96-5p and Tp73,miR-34b-3p and Cckbr targeted regulatory relationship,further verification of miR-96-5p targeted regulation of Tp73 gene expression.3.miR-96-5p could regulate the apoptosis of PC12 cell.