| Objectives:1.Predict and verify whether miR-21 binds to CRF in the prefrontal cortex.2.To explore whether prefrontal miR-21 regulates anxiety-like behavior through CRF/CRF-R1 signaling pathway.Methods:1.Target Scan、miRDB、micro RNA.org bioinformatics website predicts the top 20~50 miRNAs that may bind to CRF;C57 male mice were randomized divided into two groups : Control group,Stress group.Bilateral prefrontal tissues were taken,the relative expression level of predicted miRNA was detected by q RT-PCR,the miRNA combined with CRF was confirmed by double luciferase reporter gene assay,and the binding site of miR-21 and CRF was predicted by Targetscan bioinformatics website;Then,q RT-PCR was used to detect the expression level of CRF in PFC of mice after acute stress and the expression level of miR-21 in PFC,HIP and AM after acute stress;The expression of miR-21 in mice with or without acute stress was detected by fluorescence in situ hybridization.2.All mice were randomly divided into three groups : miR-21 agomir group,miR-21 antagomir group and control group.Agomir(20 nmol/L,1 μL)、antagomir(20 nmol/L,1μL)and saline(1 μL)were intra-PFC administrated.Mice were killed on the 3th day after injection;the overexpression and inhibition efficiency of miR-21 was detected by q RT-PCR,and the expression of CRF protein was further detected by WB;C57 male mice were randomly divided into two groups : miR-21 agomir group and control group.Agomir(20 nmol/L,1 μL)and saline(1 μL)were intra-PFC administrated.After 3~5days,the open field and elevated plus maze behavioral experiments were carried out.The interval between the two behavioral experiments was one day.3.All mice were randomly divided into three groups : miR-21 antagomir group,miR-21 antagomir + CRF-R1 antagonist group and control group.Mi R-21 antagomir(20nmol/L,1 μL)and saline(1 μL)were injected into unilateral prefrontal cortex.All mice were implanted with cannula on one side.After 3~5 days,the open field and elevated plus maze behavioral experiments were carried out.Thirty minutes before the beginning of each behavioral experiment,miR-21 antagomir + CRF-R1 antagonist group was injected with Antalarmin(1 mg/m L,0.2 μL),and the control group and miR-21 antagomir group were injected with the same volume of saline;C57 male mice were randomly divided into two groups: miR-21 antagomir group and control group.Antagomir(20nmol/L,1 μL)and saline(1 μL)were intra-PFC administrated.The expressions of β-arrestin2 、 p-PKA substrate and CREB were detected by WB.The spontaneous excitatory postsynaptic currents(s EPSCs)of pyramidal neurons were recorded by patchclamp electrophysiology,observing the changes of frequency and amplitude of postsynaptic currents.Results:1.Mi R-21 binds to CRF and is down regulated in PFC of acute restraint stress miceTarget Scan、miRDB、microRNA.org websites predicted 10 miRNAs potentially binding to CRF.The q RT-PCR results showed that the relative m RNA expression levels of miR-129,miR-429,miR-498,miR-410,miR-21 and miR-3075 of stress group were significantly decreased compared with control group(P<0.05);The results of dual luciferase reporter assay showed that the luciferase activity of miR-21 + CRF group was significantly decreased compared with miR-21 + CRF mut group(P<0.001).q RT-PCR results showed that the m RNA expression level of CRF in PFC region was significantly up-regulated compared with control group(P=0.033),and the m RNA expression level of miR-21 was significantly down-regulated compared with control group in PFC,HIP and AM(P<0.05).Fluorescence in situ hybridization showed that the expression of miR-21 in PFC region of stress group was significantly lower than that in control group.2.Low expression of miR-21 reduced anxiety-like behavior in miceq RT-PCR results showed that compared with control group,the expression of miR-21 in miR-21 agomir group increased significantly(P=0.02)and the expression of miR-21 in miR-21 antagomir group decreased significantly(P=0.007).WB results showed that the expression of CRF protein in miR-21 agomir group was significantly decreased as compared with control group(P<0.05)and that in miR-21 antagomir group increased significantly(P<0.05).The results of open field experiment showed that the time of miR-21 agomir group in the central area was significantly higher compared with control group(P=0.001),and there was no difference in the total distance between the two groups(P>0.05).The results of elevated plus maze test showed that miR-21 agomir group increased the time in mice travelled in open arms(P=0.034),and there was no difference in velocity between the two groups(P>0.05).3.Mi R-21 activates PKA signal through CRF / CRF-R1 pathway to regulate anxiety like behaviorThe results of open-field experiment showed that compared with control group,the time in the central area was significantly reduced in miR-21 antagomir group(P=0.014),while the time of miR-21 antagomir + CRF-R1 antagonist group in the central area was significantly higher than that of miR-21 antagomir group(P=0.04),and there was no difference in the total distance among three groups(P>0.05).The results of elevated plus maze test showed that compared with the control group,the open arm time of miR-21 antagomir group was significantly lower(P=0.03),while compared with miR-21 antagomir group,the open arm time of miR-21 antagomir + CRF-R1 antagonist group was significantly higher(P=0.009),and there was no difference in velocity among three groups(P>0.05).WB analysis showed that compared with control group,the expression of p-PKA substrate protein of miR-21 antagomir group decreased significantly in the prefrontal cortex(P<0.01),the expression of CREB protein increased significantly(P<0.05),while there was no significant difference in β-arrestin2 protein expression(P>0.05).Patch clamp results showed that the amplitude and frequency of the spontaneous excitatory postsynaptic current in miR-21 antagomir group were significantly higher compared with control group(P<0.05).Conclusions:1.miR-21 in the prefrontal cortex regulates the expression of CRF and anxiety-like behavior.2.Prefrontal cortex miR-21 regulates anxiety-like behavior through CRF-R1 and PKA related signaling pathway. |
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