| Post-stroke depression (PSD) is one of the most common psychological behavior disorders complications after stroke. The incidence of PSD is about25%to79%of stroke patients. The specific mechanism of PSD is not entirely clear, causing great difficulties in disease remedy. Recent research has suggested that miRNAs likely play an important role in the occurrence and development of depression, and show potential as targets for treatment of clinical depression. However, it is not yet known if miRNAs are associated with post-stroke depression. Recently, Scholars showed altered expression of various miRNAs in pe-ripheral blood, from acute stage stroke patients. A further study confirmed corresponding miRNA changes in the brain, in a mouse ischemia model. Interestingly, there are small overlaps between downregulated miRNA expression after cerebral ischemia, and in depression patients. Nevertheless, the effects of these overlapping miRNAs on the occurrence and development of post-stroke de-pression remains unclear. Therefore, in this study, we used a rat model of post-stroke depression to investigate the effects of miR-137on behavior.Part1:The establishment of post-stroke depression rats modelObjective:To establish rats model of post-stroke depression for providing experimental basis of disease research.Methods:SD rats were randomly divided into three groups:control group, stroke group and PSD. Cerebral ischemia reperfusion model was produced using the brain artery occlusion method (middle cerebral artery occlusion, MCAO). And MCAO models of Longa grade1-4were choosed to establish PSD model by chronic mild stimulation (chronic mild stress, CMS). Sugar water consumption test, the wilderness test (open-field test, OFT) and forced swimming were used respectively to assess the sensation loss, decreased activityand behavioral despair of rats. And all of the rats were sacrificed to detect the neurotransmitters levels in the hypothalamus.Results:Compared with control group, the growth of body weight of rats were obviously lagging in PSD group. At7d, PSD group rats weight were lower than the control group (P<0.05) and lower than the stroke group in14d (P<0.05). As time changes, the sucrose water consumption proportion of PSD group rats gradually declined. Compared with control group in14d and21d, sucrose water consumption of rats in PSD group was decreased obviously (P<0.05), and in21d sucrose water consumption ratio of PSD rat was lower than the stroke group (P<0.05). Change over time, the PSD group rats score level gradually decline, to21d was still declining, and scores are less than the control group at each time (P<0.05). In the21d, NE, DA and5-HT levels of the PSD group rats were significantly lower than the control group (P<0.05).Conclusion:PSD rat model could fully performant core symptoms of PSD such as lacking of sensation, decreased activity and etc. The maneuverability and repeatability of rat model is good and the rat model is an ideal model for investigation of PSD.Part2:miR-137in PSD expression in rats and its therapeutic effectObjective:To observe the expression of miRNAs in PSD rat and its effect on the treatment of PSD rat.Methods:①MCAO rats, PSD rats and normal rats were used to investigate microRNA expression in the brain via microarray detection, and the results were verificated by qRT-PCR.②Subsequently,24SD rats were divided into4groups, specifically, model, stroke, agomir-137, agomir-NC (negative control). The lasttwo groups were received an injection of agomir-137or agomir-NC into the left lateral ventricle48hours before model induction.21days later, behavioral tests of each group rats were examined.Results:①Microarray detection results showed that compared with normal rats, the levels of a variety of miRNAs expressed in brain of PSD rat increased or decreased, such as miR-137, miR-124, miR-181, miR-145, and etc. QRT-PCR verification results were consistent with the microarray results. The expression level of miR-137in the peripheral blood and brain of PSD rat were significantly lower than normal rats (p<0.05).②Wilderness, according to the results of experiment on cerebral ischemia3w, the PSD rat ventricle injection agomir-137vertical and horizontal score score were significantly higher than that of agomir-NC group of rats and normal PSD rats (p <0.05). The experimental results showed that the sugar water consumption after cerebral ischemia, at the end of the2w agomir-137percentage group rats sugar water consumption is significantly higher than agomir-NC group of rats and normal PSD rats (p<0.05).Conclusion:①Many miRNAs may participate in the pathological processes of PSD and the effect of miR-137was more noticeable.②Artificially enhanced the expression of endogenous miR-137could play a role of resistance to the PSD.Part3:miR-137by inhibiting Grin2A expression of play in the treatment of post-stroke depression model ratsObjective:In order to search the targets of miR-137involving in the treatment of PSD.Methods:①To investigate the mechanism underlying miR-137behavioral improvements in rats with post-stroke depression, potential miR-137targets were identified using the databases, pictar, microbase and targetscan.②To determine if Grin2A participates in miR-137-induced behavioral improvements in post-stroke depression rats, we injected a plasmid containing the Grin2A gene into the brain of post-stroke depression rats, via the brain ventricles.③To determine if the identified sequence downregulates Grin2A mRNA translation following miR-137binding, Grin2A3’UTRs containing the target site (Grin2A-3’UTR-wt) or a mutated site (Grin2A-3’UTR-mut), were constructed in a luciferase reporter system. Subsequently, miR-137and either luciferase empty vector or Grin2A-3’UTR-wt or Grin2A-3’UTR-mut plasmids were co-transfected into HEK-293cells. HEK-293cells were incubated in RPMI1640medium containing10%fetal bovine serum at37℃in an incubator containing5%CO2. Full-length Grin2A3’UTR containing the miR-137binding site, was PCR amplified and cloned into HindⅢ and SacⅠ sites of the pMIR-REPORT miRNA expression reporter vector. The resulting vector was named Grin2A-3’UTR-wt. A point mutation of Grin2A-3’UTR-wt was identified using the Easy Mutagenesis System and subcloned also, with the resulting vector named Grin2A-3’UTR-mut. HEK293T cells were seeded into24-well plates and divided into three groups. Empty vector (400ng) was added to the control group, Grin2A-3’UTR-wt plasmid (400ng) to the wild-type group, and Grin2A-3’UTR-mut plasmid (400ng) to the mutation group. Plasmids and miR-137were transfected using Lipofectamine2000, and20ng pRL-TK was added as an internal reference. Within each group, parallel wells were used for the addition of mimic-NC, a negative control for miR-137mimic (miR-con). After36hours of transfection, the fluorescence intensity of cells was measured using the Dual-Luciferase Reporter Assay System.④24rats were randomly assigned to4groups, specifically, agomir-137, agomir-NC (negative control), agomir-137+Grin2A and agomir-137+vector groups. The four agomir groups received an injection of agomir-137, a miR-137antagonist into the left lateral ventricle48hours before model induction. Agomir-NC, LV-CMV-Grin2A and-control plasmids were also injected into the left lateral ventricle48hours before model induction.Results:①Moreover, western blot assays showed that in PC12cells, miR-137mimic (mimics of synthesized mir-137, showing similar effects to natural mir-137) significantly reduced (P<0.05), but miR-137inhibitor significantly increased (P<0.05), Grin2A levels. These results suggest that miR-137binds to the3’UTR of Grin2A mRNA and regulates Grin2A protein expression.②Luciferase assays found significantly lower fluorescence intensities in HEK-293cells transfected with Grin2A-3’UTR-wt, compared with negative controls (miR-con)(P<0.05). However, no significant difference was detected between HEK-293cells transfected with Grin2A-3’UTR-mut or empty vector (P>0.05; Figure3B). This suggests miR-137binds to the3’UTR of Grin2A mRNA, and regulates its translation.③We found significantly lower locomotor and rearing activities in the agomir-137+Grin2A group than in the negative control (agomir-137+vector) and agomir-137groups (P<0.05). Moreover, the sucrose consumption percentage was significantly lower in the agomir-137+Grin2A group than in the negative control and agomir-137groups (P<0.05). These results suggest that Grin2A overexpression prevents the improved behavioral effects of miR-137in rats with post-stroke depression, and confirms the involvement of Grin2A in the miR-137therapeutic effect in post-stroke depression rats..Conclusion:miR-137could bind with Grin2A mRNA3’UTR and downregulated its protein expression at the post-transcriptional level. Overexpression of Grin2A could block theinfluence of mi-137on ethology of PSD rat.The conclusions:1. PSD rat model could fully performant core symptoms of PSD such as lacking of sensation, decreased activity and etc. The maneuverability and repeatability of rat model is good and the rat model is an ideal model for investigation of PSD.2. Many miRNAs may participate in the pathological processes of PSD and the effect of miR-137was more noticeable. Artificially enhanced the expression of endogenous miR-137could play a role of resistance to the PSD. 3. miR-137could bind with Grin2A mRNA3’UTR and downregulated its protein expression at the post-transcriptional level. Overexpression of Grin2A could block theinfluence of miR-137on ethology of PSD rat. |
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