The Role And Underlying Mechanism Of MiR-214-3p In Depressive-like Behaviors Of Mice Induced By Chronic Social Defeated Stress

Part I The changes in the expression of miRNAs and β-catenin induced by chronic social defeated stress in prefrontal cortex of miceObjective:To establish the model of chronic social defeated stress(CSDS) and assay the proteins expression of β-catenin and miRNAs.Methods:To establish the major depression models by chronic unpredicted mild stress (CUMS) and CSDS. To identify the model behaviors by sucrose preference test, tail suspended test and social interaction test. To detect corticosterone concentration of mice serum by ELSA. Quantitative real-time PCR experiments were performed to quantitative analyze differential expression of microRNAs in prefrontal cortex of mice. Western blotting was used to detected protein expression of P-catenin in prefrontal cortex.Results:(1) The expressions of miR-214-3p and miR-690in prefrontal cortex of CSDS mice up-regulated to4.249±0.816and2.527±0.292, respectively when compared to control.(2) The sucrose preference ratio of CSDS mice was decreased to40.577±4.81%, the interaction time in social interaction test was decreased to60.85±13.17s.(3) Corticosterone concentration in CSDS mice serum increased to17.943±0.86ug/L.(4) The expression of β-catenin in CSDS mice was decreased to54.7±11.2%.Conclusion:CSDS and CUMS are ideal models for major depression research. The expression of miR-214-3p is up-regulated and the expression β-catenin is decreased in prefrontal cortex of CSDS. Part II The study of miR-214-3p and miR-690targetsObjective:To conform that the targets of miR-214-3p and miR-690were ctnnblMethods:Quantitative real-time PCR experiments were performed to quantitative analyze expressions of miR-214-3p and miR-690in prefrontal cortex of mice. Western blotting was used to detected protein expression of β-ctenin in prefrontal cortex. Dual-luciferase reporter assay system was used to demonstrate the3’UTR of ctnnbl mRNA is the target of miR-690.Results:(1) The expression of miR-690was increased to44.2±0.44after transfecting the mimic of miR-690in neurons compared to control.(2) The expression of β-catenin was decreased to60±12%after transfecting the mimic of miR-690in neurons when compared to control.(3) Dual luciferase report experiment demonstrated that3’UTR of ctnnbl was the target of miR-690.(4) Injection the mimics of miR-214-3p and miR-690in vivo by the3th ventricle increased the expressions of miR-214-3p and miR-690to2.456±0.032and1.21±0.05respectively.(5) Injection the mimics of miR-214-3p in vivo by the3th ventricle decreased the expression of P-catenin to64.6±4.35, but miR-690had no effect on the expression of β-catenin.(6) Silencing the3’UTR of ctnnbl in vivo decreased the expressions of mRNA and β-catenin to0.37±0.07and24±3.5%, respectively.Conclusion:3’UTR of ctnnbl is the target of miR-214-3p and miR-214-3p regulates the protein expression. Part Ⅲ The effect and mechanism of miR-214-3p on depressive-like behaviors of mice induced by chronic social defeated stressObjective:To study the effect and mechanism of miR-214-3p on depressive-like behaviors in prefrontal cortex induced by CSDSMethods:Sucrose preference test, tail suspended test and social interaction test were used after inhibitor injection by3th ventricle in CSDS mice. Quantitative real-time PCR experiments were performed to quantitative analyze expressions of miR-214-3p.Western blotting was used to detected protein expression of β-ctenin in prefrontal cortex. Slice patch clamp was used to record mEPSC.Results:(1) Antagomir-214decreased the expression of miR-214-3p to1.046±0.05and increased the expression of β-catenin to91.3±4.7%.(2) Antagomir-214decreased the immobility time to27.954±4.06s in tail suspended test and increased the interaction time in social interaction test to64.283±10.7s.(3) Antagomir-214increased the amplitude of mEPSC in prefrontal cortex of CSDS mice.(4) Lv-miR-214decreased the expression of miR-214-3p to1.013±0.046and increased the expression of β-catenin to227q59.8%.(5) Lv-miR-214decreased the immobility time to45.7±12.08s in tail suspended test and increased the interaction time to53.6±10.51s in social interaction test.(6) Over-expression of ctnnbl decreased the immobility time to37.5±6.91s in tail suspended test and increased the interaction time to46.883±6.921s in social interaction test.(7) Intranasal delivery of antagomir-214decreased the expression of miR-214-3p to0.4q0.1and increased the expression of β-catenin to110q9.3%respectively compared to control.(8) Intranasal delivery of antagomir-214decreased the immobility time to67.83q14.35s in tail suspended test and increased the interaction time to50.55q7.44s in social interaction test.(9) Antagomir-214, LV-miR-214and over-expression of ctnnbl had no effects on the sucrose preference ratio of CSDS mice.Conclusion:Ihibition of miR-214-3p inhibitor antagomir-214reverses depressive-like behaviors of CSDS mice.