miR-205-3p在氧糖剥夺-复氧复糖星形胶质细胞胀亡中的作用:与AQP4的关系

Role of miR-205-3p in oncosis in astrocytes subjected to oxygen-glucose deprivation and restoration: relationship with AQP4

目的评价miR-205-3p在氧糖剥夺-复氧复糖(OGD/R)星形胶质细胞胀亡中的作用及其与水通道蛋白4(AQP4)的关系。方法体外培养原代星形胶质细胞至对数生长期,采用随机数字表法分为5组(n=16):对照组(C组)、OGD/R组(O组)、OGD/R+miR-205-3p模拟物组(M组)、OGD/R+miR-205-3p抑制剂组(I组)和OGD/R+阴性对照组(NC组)。C组在正常条件下培养,O组于37℃厌氧孵育箱(含94%N_(2)、1%O_(2)和5%CO_(2))氧糖剥夺4 h,于复氧复糖24 h;M组、I组和NC组分别转染miR-205-3p模拟物、抑制剂和阴性对照后,氧糖剥夺4 h,复氧复糖24 h。采用CCK-8法检测细胞活力,流式细胞术分析细胞损伤及胀亡情况,qRT-PCR法检测AQP4 mRNA表达,Western blot法检测AQP4及porimin的表达。结果与C组相比,O组miR-205-3p表达下调,细胞活力降低,细胞损伤率及胀亡率升高,AQP4及其mRNA和porimin表达上调(P<0.05);与O组相比,M组miR-205-3p表达上调,细胞活力升高,细胞损伤率及胀亡率降低,AQP4及其mRNA和porimin表达下调,I组miR-205-3p表达下调,细胞活力降低,I组细胞损伤率及胀亡率升高,AQP4及其mRNA和porimin表达上调(P<0.05),NC组差异无统计学意义(P>0.05)。结论miR-205-3p参与了OGD/R星形胶质细胞胀亡的过程,与调控AQP4表达有关。

Objective To evaluate the role of miR-205-3p in oncosis in astrocytes subjected to oxygen-glucose deprivation and restoration(OGD/R)and the relationship with aquaporin4(AQP4).Methods Primary astrocytes were cultured in vitro to the logarithmic growth phase and divided into 5 groups(n=16 each)using a random number table method:control group(C group),OGD/R group(O group),OGD/R+miR-205-3p mimic group(M group),OGD/R+miR-205-3p inhibitor group(I group),and OGD/R+negative control group(NC group).Cells were cultured routinely in C group.Cells were subjected to 4 h of oxygen-glucose deprivation in a 37℃anaerobic incubator(containing 94%N_(2),1%O_(2) and 5%CO_(2))followed by restoration of O_(2)-glucose supply for 24 h in O group.Cells in M,I and NC groups were transfected with miR-205-3p mimic,miR-205-3p inhibitor and miR-205-3p negative control for 48 h,respectively,and then cells were subjected to 4 h of oxygen-glucose deprivation followed by restoration of O_(2)-glucose supply for 24 h.The cell viability was evaluated by CCK-8 assay,the cell injury and oncosis were analyzed by flow cytometry,the expression of AQP4 mRNA was detected by quantitative reverse transcription-polymerase chain reaction,and the expression of AQP4 and porimin was detected by Western blot.Results Compared with C group,the expression of miR-205-3p was significantly down-regulated,the cell viability was decreased,the rates of cell injury and oncosis were increased,and the expression of AQP4 protein and mRNA and porimin was up-regulated in O group(P<0.05).Compared with O group,the expression of miR-205-3p was significantly up-regulated,the cell viability was increased,the rates of cell injury and oncosis were decreased,and the expression of AQP4 protein and mRNA and porimin was down-regulated in M group,the expression of miR-205-3p was significantly down-regulated,the cell viability was decreased,the rates of cell injury and oncosis were increased,and the expression of AQP4 protein and mRNA and porimin was up-regulated in I group(P<0.05),and no significant changes were found in NC group(P>0.05).Conclusions miR-205-3p is involved in oncosis in astrocytes subjected to OGD/R,which is associated with regulation of AQP4 expression.