Preliminary Study On The Role And Mechanism Of MiR-711 In Alzheimer's Disease

Objective1.To investigate the expression levels of miR-711 in peripheral blood of patients with AD and normal controls,and discuss their clinical significance.2.To investigate the role and mechanism of miR-711 in the AD cell model induced by A?_1-40.Methods1.Thirty Normal Controls?NC?and forty patients with Alzheimer's disease were enrolled in this study.The expression levels of serum miR-711 were detected by real-time fluorescent quantitative PCR?qRT-PCR?.In addition,the relationship between the expression levels of miR-711 and the Montreal Cognitive Evaluation Scale?MoCA?were analyzed.The differences between serum miR-711 in patients with different severity of AD were also explored.Finally the ROC curve was used to evaluate the diagnostic value of serum miR-711 for AD.2.The concentration gradient of A?_1-40-40 was set and used to treated SH-SY5Y cells.After 24 hours of culture,the cell inhibition rate was measured by CCK8 kit and selected the optimal concentration for constructing the AD cell model.Next,AD cells were then transfected with miR-711 mimics,miR-711 inhibitor,and negative controls mediated by Lipo2000.Then qRT-PCR was used to detect the difference in miR-711expression levels between the blank group?only SH-SY5Y cells?,model group,miR-711 mimics group,miR-711 inhibitor group and negative control group.Finally,the cell survival rate of each group was detected by the CCK8 kit and the expression levels of Akt,Bcl-2 and Caspase-3 mRNA were estimated by qRT-PCR.Results1.Serum miR-711 in AD patients were increased significantly compared with NC group?P<0.001?.Besides,the expression levels of miR-711 in serum were negatively correlated with the MoCA scores according to correlation analysis?r=-0.457,P<0.001?.2.According to the Clinical Dementia Rating Scale?CDR?scores,the severity of the disease was evaluated,and AD patients were separated into three groups:mild group,moderate group and severe group.There were differences in the expression of miR-711 among three groups'serum?P<0.05?.The levels of serum miR-711 in the severe AD group were increased in contrast with other groups?P<0.05?.3.When serum miR-711 was used to diagnose AD,the AUC was 0.841.4.A?_1-40-40 treated SH-SY5Y cells?15umol/L,24 hours?could construct a successful of AD model.In addition,the concentration of A?_1-40-40 was proportional to the inhibition rate of cells.5.Compared to blank group,the expression levels of miR-711 in model group were increased?P<0.01?.And the levels of miR-711 transfection with miR-711mimics were significantly higher than in model group?P<0.001?,and transfection with miR-711 inhibitor could inhibit its expression?P<0.05?,while the negative control group had no significant effect on the expression levels of miR-711?P>0.05?.6.In contrast with blank group,the cell survival rate in model group was significantly reduced?P<0.001?,and was about a half inhibition rate.The cell survival rate in miR-711 mimics group was significantly declined in comparison with model group?P<0.01?,then knocking down the miR-711 would mitigate cell inhibition?P<0.01?,while the cell viability in the negative control group was not significantly affected?P>0.05?.7.Compared to the blank group,Akt and Bcl-2 mRNA were down-regulated in model group?P<0.01;P<0.05?,while the levels of Caspase-3 mRNA raised?P<0.05?.Over-expression of miR-711 reduced Akt and Bcl-2 mRNA levels,and Caspase-3mRNA levels increased more remarkably compared with model group?P<0.05;P<0.05;P<0.01?,but the results of knocking down miR-711 were the opposite?P<0.01;P<0.01;P<0.05?.However,there was no significant change of three mRNAs in negative control group.Conclusions1.The expression levels of serum miR-711 in AD patients were beyond than in NC,and it was related to the level of cognition and the severity of the disease,which can be used as a potential clinical diagnostic biomarker of AD.2.Over-expression of miR-711 could inhibite cell growth of AD,meanwhile it could down-regulate Akt and Bcl-2,up-regulate Caspase-3 to activate apoptotic pathways or regulate the expression of apoptosis-related genes,inhibiting its expression can improve the inhibition of AD cells and block the apoptotic pathway.This provides in vitro cytological evidence for potential therapeutic targets of AD.