Effects Of Dexamethasone On MiR-183-5p Expression In Fetal Rat Cerebral Cortex Neurons In Vitro

Objective: To investigate the effects of Dexamethasone(DEX)on the expression of mi R-183-5p and GR protein in cultured fetal rat cortical neurons in vitro,verify the mi R-183-5p to be involved in regulating the expression of GR protein encoded by Nr3c1 gene,and clarify the possible mechanism of the effect of glucocorticosteriod(GCs)on developing neurons.Methods:1.Primary fetal rat cortical neurons were cultured in vitro and cell models of dexamethasone intervention on fetal cortical neurons were made.According to the different final concentration of dexamethasone,they were divided into control group(0μmol/L)and intervention group(10μmol/L).Quantitative PCR was used to detect the effects of dexamethasone on the expression of rno-mi R-183-5p and Nr3c1 m RNA in cultured cortical neurons on 3 and 7 days of intervention.Western blot analysis was used to detect the effect of dexamethasone on GR protein expression in cultured cortical neurons in vitro.2.Plasmid p HG-Mir Target-rat Nr3c1-3’UTR-WT and p HGMir Target-rat Nr3c1-3’UTR-mut were constructed by site-directed mutation of the specific binding region base of Nr3c1 gene.According to transfection plasmid and mi RNA,the plasmid was divided into 4groups: Nr3c1-3’UTR-WT negative control group(NC),Nr3c1-3’ UTRWT/mi R-183-5p mimic group,Nr3c1-3’UTR-mut NC group,and Nr3c1-3’UTR-MUT/mi R-183-5p mimic group.The regulation of Nr3c1 m RNA expression by rno-mi R-183-5p was detected by dual luciferase reporting assay.3.Using liposome transfection method,rno-mi R-183-5p mimics,rno-mi R-183-5p inhibitor and corresponding negative control NC were instantaneously transfected into 7d primary cultured fetal rat cerebral cortex neurons,which were divided into 4 groups according to the transfected mi RNA: rno-mi R-183-5p mimics NC group,rno-mi R-183-5p mimics group,rno-mi R-183-5p inhibitors NC group,and rno-mi R183-5p inhibitors group.Quantitative PCR was used to detect the influence of overexpression/inhibition of rno-mi R-183-5p on Nr3c1 m RNA expression,and western blot was used to detect the influence of overexpression/inhibition of rno-mi R-183-5p on GR protein expression.Results:1.Quantitative PCR showed that the expression of mi R-183-5p in the intervention group was higher than that in the control group after dexamethasone intervention for 3 days,and the difference was statistically significant(P<0.05),Nr3c1 m RNA expression was significantly different between the two groups(P<0.05),Nr3c1 m RNA expression in the intervention group was lower than that in the control group,and the difference was statistically significant(P<0.05);After 7days of intervention,there was no significant difference in mi R-183-5p and Nr3c1 m RNA expression between the two groups(P>0.05).Western blot showed that there was no significant difference in Nr3c1 protein expression in cultured neurons between the two groups after dexamethasone intervention for 3 days(P>0.05);After 7 days of intervention,Nr3c1 protein expression in the intervention group was lower than that in the control group,and the difference was statistically significant(P<0.05).2.After the luciferase reporter gene was co-transfected into fetal rat cortical neurons,the relative fluorescence values of each group were significantly different(P<0.05),the relative fluorescence values of Nr3c1-3’UTR-WT/mi R-183-5p mimic group were significantly lower than those of the other three groups(P<0.05).3.Quantitative PCR and western blot showed that Nr3c1 m RNA and GR protein expression were down-regulated in mi R-183-5p mimic group compared with mi R-183-5p inhibitor group,and the differences were statistically significant(P< 0.05)Conclusions: Dexamethasone may negatively regulate the expression of glucocorticoid receptor gene Nr3c1 by targeting mi R-183-5p.