circRIMS2靶向miR-186调控BDNF表达减轻CRS诱发的小鼠抑郁样行为

CircRIMS2 targeting miR-186 to regulate BDNF expression and alleviate CRS induced depression like behavior in mice

目的探讨抑郁样行为小鼠体内中circRIMS2变化及circRIMS2对糖氧剥夺(OGD)诱导HT22细胞凋亡的影响。方法(1)将45只C57BL/6雌性小鼠随机分为对照组、模型组与氟西汀组,每组15只。通过慢性束缚应激(CRS)对模型组和氟西汀组小鼠构建抑郁样行为模型,对氟西汀组小鼠给予10 mg/kg的氟西汀治疗。采用糖水偏好测试、悬尾测试、强迫游泳测试和开野测试评估小鼠抑郁样行为。评估后取小鼠血清和大脑海马组织,采用逆转录实时定量聚合酶链式反应(RT-qPCR)检测环状RNA、miR-186及BDNF mRNA的表达水平。采用Western blot法检测小鼠海马组织中BDNF蛋白表达情况。(2)在HT22细胞中分别加入慢病毒载体pLO-circCon、pLO-circRIMS2转染试剂后,取一部分HT22细胞进行OGD处理以构建体外凋亡模型。根据进行过的细胞转染和OGD操作,将HT22细胞分为Control+pLO-circCon组、Control+pLO-circRIMS2组、OGD+pLO-circCon组和OGD+pLO-circRIMS2组。采用RT-qPCR检测4组细胞中circRIMS2、miR-186及BDNF mRNA的表达水平,通过流式细胞术检测并计算4组细胞凋亡率。采用Western blot法检测细胞中BDNF蛋白表达情况。结果(1)与对照组相比,模型组小鼠糖水偏好率低,悬尾与强迫游泳不动时间长,开野测试得分低,血清中circTRAM2、circTNIK与circTFRC表达水平高,circDOCK4、circRIMS2,circSTAG1表达水平低,差异均有统计学意义(均P<0.01)。与模型组相比,氟西汀组小鼠糖水偏好率高,悬尾与强迫游泳不动时间短,开野测试得分高,血清中circRIMS2表达水平高,差异均有统计学意义(均P<0.01)。(2)与Control+pLO-circCon组相比,Control+pLO-circRIMS2组细胞中circRIMS2、BDNF mRNA表达水平高,miR-186表达水平和细胞凋亡率低,差异均有统计学意义(均P<0.01)。与OGD+pLO-circCon组相比,OGD+pLO-circRIMS2组细胞中circRIMS2、BDNF mRNA表达水平高,miR-186表达水平和细胞凋亡率低,差异均有统计学意义(均P<0.01)。结论circRIMS2基因与小鼠抑郁样行为存在关联,抑郁样行为小鼠血清中circRIMS2表达降低,氟西汀治疗能提高circRIMS2表达。慢病毒质粒提高细胞中circRIMS2基因表达,能通过miR-186/BDNF信号减轻OGD诱发的神经元凋亡。

Objective To explore the changes of circRIMS2 in mice with depression like behavior and the influence of circRIMS2 on oxygen-glucose deprivation(OGD)induced apoptosis of HT22 cells.Methods(1)Forty-five C57BL/6 female mice were randomly divided into the control group,model group,and fluoxetine group,with 15 mice in each group.A depression like behavior model was constructed in both the model group and fluoxetine group mice through chronic restraint stress(CRS),and fluoxetine group mice were treated with 10 mg/kg of fluoxetine.Depression like behavior in mice was evaluated using the sucrose preference test,tail suspension test,forced swimming test,and open field test.After evaluation,mouse serum and hippocampal tissue were collected,and the expression of circular RNA,miR-186,and BDNF mRNA were detected using reverse transcription real-time quantitative polymerase chain reaction(RT-qPCR).Western blot was used to detect the expression of BDNF protein in mice hippocampus.(2)After adding lentiviral vectors pLO-circCon and pLO-circRIMS2 transfection reagents to HT22 cells,a portion of HT22 cells were subjected to OGD treatment to construct an in vitro apoptosis model.Based on the cell transfection and OGD procedures,HT22 cells were divided into Control+pLO-circCon group,Control+pLO-circRIMS2 group,OGD+pLO-circCon group,and OGD+pLO-circRIMS2 group.RT-qPCR was used to detect the expression of circRIMS2,miR-186,and BDNF mRNA in four groups of cells,and flow cytometry was used to detect and calculate the apoptosis rate of the four groups of cells.Results(1)Compared with the control group,the model group mice had a lower preference for sucrose,longer tail suspension and forced immobility time,lower open field test scores,higher expression levels of circTRAM2,circTNIK,and circTFRC in serum,and lower expression levels of circDOCK4,circRIMS2,and circSTAG1,and the differences were statistically significant(all P<0.01).Compared with the model group,the fluoxetine group mice had a higher preference for sucrose,shorter tail suspension and forced swimming immobility time,higher open field test scores,and higher level of circRIMS2 expression in serum,with statistical differences(all P<0.01).(2)Compared with the Control+pLO-circCon group,the Control+pLO-circRIMS2 group had higher levels of circRIMS2 and BDNF mRNA expression,lower level of miR-186 expression,and lower cell apoptosis rate,with statistically significant differences(all P<0.01).Compared with the OGD+pLO-circCon group,the OGD+pLO-circRIMS2 group had higher levels of circRIMS2 and BDNF mRNA expression,lower level of miR-186 expression,and lower cell apoptosis rate,with statistically significant differences(all P<0.01).Conclusions The circRIMS2 gene is associated with depression like behavior in mice.The expression of circRIMS2 in the serum of mice with depression like behavior is reduced,and fluoxetine can increase the expression of circRIMS2.The lentiviral plasmid enhances the expression of circRIMS2 gene in cells and can alleviate OGD induced neuronal apoptosis through miR-186/BDNF signaling.