Screening And Identifying Of MiRNAs In Brain Derived LncRNA That Involved In The Regulation Of Neuronal Survival

Objective: To identify miRNAs and their regulatory mechanisms in brain derived LncRNA involved in the regulation of neuronal survival.Methods: The homologous sequence alignment and possible miRNAs screening and localization of brain derived 6.1 kb LncRNA were performed Through Pubmed,miRNA databases and prediction software.Constructed candidate miRNA over-expression vector was transfected into primary cultured rat embryonic cerebral cortical neurons using liposome transfection,and then the neuronal death induction experiments were proceeded to test the changes of mortality in embryonic cortical neurons transfected with candidate miRNAs.And the express changes of the target gene was detected by qPCR and Western blotting.Results:(1)Locations and sequences of candidate miRNAs were identified by screening and prediction software.(2)Successfully constructed of candidate miRNAs over-expression vector.(3)Identification for primary cultured rat embryonic cerebral cortical neurons.(4)miR-4 showed a protective effect on neuronal survival in the induction of neuronal cell death for 48 h.Mortality rate of neuronal cell was significantly lower in miR-4 group than that in negative control group(P <0.05).(5)miR-4 showed the protective function for neurons in the neuronal cell death induction test in 48 h,the death rate was significantly lower than that of the negative control group,and the difference was statistically significant(P <0.05),which was consistent with the results of primary screening.(6)The mRNA expression levels of mapk10,Snap25,Ncan,Atp2b3,CNA2D2,INHBA,CAMTA1 and CALCOCO1 were detected by qPCR.The results showed that the mRNA expression levels of Ncan,Atp2b3,CACNA2D2 and CALCOCO1 were significantly lower in miR-4 group than negative control group(P <0.05).(7)Transfected with miR-4 over-expressing vector,the expression of BDNF was detected by qPCR.The results showed that the expression level of BDNF transfected with miR-4 over-expressing vector was significantly higher than that in negative control group(P <0.05).(8)The BDNF protein,as the downstream target gene of miR-4,was detected.The results showed that the expression level of BDNF was positively correlated with miR-4(P <0.05),and the difference was statistically significant(P <0.05).Conclusion: The mechanism of survival protection of brain derived 6.1kb LncRNA on neurons may be achieved through miR-4.miR-4 promotes the survival of neurons by the up-regulation expression of BDNF gene.