| BackgroundAlcohol dependence is a chronic and complex disease,affected by genetic,epigenetic and environmental factors.Alcohol dependence causes lasting cellular changes in the brain,and these changes may be partly caused by constant changes in gene expression.miRNA is a post-transcriptional regulator of gene expression,a negative regulator of mRNA expression,and is related to addiction-related behaviors.Studies showed that rats could induce abnormal expression of miRNAs after excessive alcohol intake,influence the formation of alcohol dependence by regulating a variety of neurotransmitter-related proteins and nutritional factors,and plays an important regulatory role in the evolution of Clinical or behavioral symptoms(e.g.,Craving,withdrawal,relapse,cognitive impairment).The striatum is a key part of the brain for addiction,and a large number of studies have shown that the nucleus accumbens is also a key part of the formation of alcohol dependence and craving.However,the changes in striatal miRNA and target genes during the formation of alcohol dependence and the evolution of clinical manifestations still need to be further studied.ObjectivesIn order to vertify the regulatory effect of miRNA on mRNA,an alcohol-dependent rat model was constructed and the differentially expressed miRNA and mRNA was screened out in the striatum of the alcohol-dependent model rat.Find out the target gene action site,and further explore the function of striatal miRNA in alcohol dependence.The target gene action site was determined and the function of striatal miRNA in alcohol dependence was further explored.Methods1.The total RNA of the striatal brain area of the alcohol-dependent rats(n=6)and the control rats(n=6)was extracted,and miRNA and mRNA sequencing were performed by high-throughput sequencing methods,and differentially expressed genes were screened out.Informatics methods was used to analyze the relationship between miRNA and mRNA and construct a miRNA-mRNA negative regulatory network.2.Differentially expressed mRNAs was screened out in the brain regions of the striatum of alcohol-dependent rats using databases such as NCBI and GCBI.MiRNA was predicted and MiRNA-mRNA was screened using miRWalk and other software based on the results of differentially expressed mRNAs and the constructed miRNA-mRNA negative regulatory network.3.A new CPP model was constructed.The striatum tissue was collected for the following verification test.4.Real-time fluorescent quantitative PCR technology was used to verify the expression of the screened mRNA and miRNA at the tissue level.3.Construct a CPP model and take the striatum tissue for use.4.At the tissue level,real-time fluorescent quantitative PCR technology is used to verify the expression of the screened mRNA and miRNA.5.The targeted regulation of miRNA on mRNA was verified at the cellular level by intervening in PC12 cells miRNA mimic/inhibitor using the real-time fluorescent quantitative PCR technology.6.Luciferase reporter gene experiment was conducted to using dual luciferase reporter gene experiment to detect rno-miR-26b-5p to Scn5a,rno-miR-455-3p to Scn5a,rno-miR-181b-5p to Cxcl10,rno-miR-298-5p to Cxcl10,rno-miR-29c-3p to Pcdh11x,rno-miR-582-3p to the 3’untranslated region of Pcdh11x,verify rno-miR-26b-5p to Scn5a,rno-miR-455-3p to Scn5a,rno-The targeted regulation of miR-181b-5p on Cxcl10,rno-miR-298-5p on Cxcl10,rno-miR-29c-3p on Pcdh11x,and rno-miR-582-3p on Pcdh11x.Results1.Ten significantly different miRNA and nine mRNA genes was screened out using high-throughput sequencing results of striatum brain regions of alcohol-dependent and control rats.,of which 4 were significantly up-regulated and 6were significantly down-regulated.Further analysis was performed on the sequencing results of mRNA and miRNA to construct a miRNA-mRNA negative regulatory network(both P<0.05).2.The targeted genes,related to alcohol dependence,substance addiction,neuron protection,neuroinflammation,neuronal growth and apoptosis,synaptic development and remodeling(i.e.,Scn5a,Pcdh11x,Cxcl10 and rno-miR-29c-3p,rno-miR-582-3p,rno-miR-298-5p,rno-miR-26b-5p,rno-miR-455-3p,rno-miR-181b-5p),was screened out.The real-time fluorescent quantitative PCR was performed on the alcoholic dependence and control rats,and the real-time fluorescent quantitative PCR results was consistend with the high-throughput sequencing results(P<0.05).regulation3.The expression level(up-regulation,or down-regulation)of miRNA in the striatum brain area of alcohol-dependent rats was simulated by interfering cells with miRNA mimic/inhibitor.The real-time fluorescent quantitative PCR results showed that Scn5a and rno-miR-26b-5p,Scn5a And rno-miR-455-3p;Cxcl10 and rno-miR-181b-5p,Cxcl10 and rno-miR-298-5p;Pcdh11x and rno-miR-29c-3p,Pcdh11x and rno-miR-582-3p for alcohol Rely on the differentially expressed miRNA-mRNA negative regulatory pair in rat striatum.Preliminarily confirmed the targeted regulation of miRNA-mRNA in the above 6 groups(all P<0.05).2.Screen out genes that may be related to alcohol dependence,substance addiction,neuron protection,neuroinflammation,neuronal growth and apoptosis,synaptic development and remodeling,such as Scn5a,Pcdh11x,Cxcl10 and rno-miR-29c-3p,rno-miR-582-3p,rno-miR-298-5p,rno-miR-26b-5p,rno-miR-455-3p,rno-miR-181b-5p,and in alcoholic dependence In rat striatum,the gene expression level was verified,and the real-time fluorescent quantitative PCR experiment results were basically consistent with the sequencing results(P<0.05)3.Use miRNA mimic/inhibitor to interfere with cells,simulate the expression level of miRNA in the striatum brain area of alcohol-dependent rats,up-regulate/down-regulate miRNA,real-time fluorescent quantitative PCR results show: Scn5a and rno-miR-26b-5p,Scn5a And rno-miR-455-3p;Cxcl10 and rno-miR-181b-5p,Cxcl10 and rno-miR-298-5p;Pcdh11x and rno-miR-29c-3p,Pcdh11x and rno-miR-582-3p for wine Rely on the differentially expressed miRNA-mRNA negative regulatory pair in rat striatum.Preliminarily confirmed the targeted regulation of miRNA-mRNA in the above 6 groups(all P<0.05).4.The fluorescence of the wild-type vector r-Pcdh11x-WT group was significantly decreased after transfection of rno-miR-29c-3p mimic.However,the decrease of the fluorescence in the mutant vector r-Pcdh11x-MUT was significantly reduced after mutation of the predicted rno-miR-29c-3p target site.After transfection of rno-miR-181b-5p inhibitor,the reporter fluorescence of the wild-type vector r-Cxcl10-WT group was significantly increased.However,the fluorescence of the mutant vector r-Cxcl10-MUT was decreased after the predicted target site was mutated..The fluorescence of the wild-type vector r-Cxcl10-WT group was significantly increased after transfection with rno-miR-298-5p inhibitor.However,the fluorescence of the mutant vector r-Cxcl10-MUT was decreased after the predicted target site was mutated.After transfection with rno-miR-455-3p inhibitor,the fluorescence of the wild-type vector r-Scn5a-WT group was significantly increased.However,the fluorescence of the mutant vector r-Scn5a-MUT was decresed after the predicted target site was mutated.Conclusions1.In the brain region of the striatum of CPP model rats,the Cxcl10 and Scn5a genes are up-regulated,while Pcdh11x genes are down-regulated.The rno-miR-29c-3p and rno-miR-582-3p are up-regulated and rno-miR-26b-5p,rno-miR-455-3p,rno-miR-181b-5p,rno-miR-298-5p are down-regulated.2.Targeted relationship of rno-miR-26b-5p to Scn5a,rno-miR-455-3p to Scn5a,rno-miR-181b-5p to Cxcl10,rno-miR-298-5p to Cxcl10,rno-miR-29c-3p targets Pcdh11x,rno-miR-582-3p targets Pcdh11x was obtained.3.It is testified that rno-miR-29c-3p,rno-miR-181b-5p,rno-miR-298-5p and rno-miR-455-3p was the translation region regulatory site,respectively. |
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