摘要
目的探讨miR-22-3p在氧-糖剥夺/复氧(OGD/R)心肌细胞铁死亡中的作用及机制。方法大鼠心肌细胞系H9c2采用OGD/R处理以及转染miR-22-3p模拟物(miR-22-3p mimic)。细胞根据是否缺氧及有无转染分为常氧组、OGD/R组、OGD/R+miRNA模拟物阴性对照(miRNA-NC组)和OGD/R+miR-22-3p组。采用细胞计数(CCK-8)法检测细胞活力,酶联免疫吸附测定(ELISA)试剂盒检测H9c2细胞中铁死亡指标水平,Western blot检测谷胱甘肽过氧化物酶4(Gpx4)及多形性腺瘤基因样蛋白2(PLAGL2)蛋白表达,双荧光素酶报告实验验证miR-22-3p和PLAGL2靶向关系。结果OGD/R处理H9c2细胞后,第4天的吸光度值低于常氧组,活性氧(ROS)及丙二醛(MDA)水平、Fe^(2+)的积累明显高于常氧组,miR-22-3p水平明显低于常氧组(t分别=3.78、4.24、2.54、4.47、3.53,P均<0.05),H9c2细胞转染miR-22-3p mimic及OGD/R处理后,第3、4天的吸光度值高于miR-NC+OGD/R组(t分别=2.98、1.97、2.57、2.46,P均<0.05),细胞MDA、ROS、Fe^(2+)明显低于OGD/R+miR-NC组(t分别=2.62、5.35、4.11,P均<0.05),Gpx4表达水平高于OGD/R+miR-NC组。Fer-1和OGD/R处理H9c2细胞后,第3、4天的细胞吸光度值明显高于OGD/R组(t分别=3.12、3.36,P均<0.05),细胞MDA、ROS和Fe^(2+)水平显著低于OGD/R组(t分别=3.03、5.33、2.48,P均<0.05)。H9c2细胞转染miR-22-3p组的PLAGL2蛋白表达明显低于miR-NC组,且PLAGL2-WT+miR-22-3p组的荧光素酶活性明显低于PLAGL2-MUT+miR-22-3p组(t=2.69,P<0.05)。H9c2细胞共转染miR-22-3p mimic和PLAGL2过表达质粒后,Gpx4表达低于OGD/R+miR-22-3p组,吸光度值明显低于OGD/R+miR-22-3p组(t分别=3.91、3.12,P均<0.05),MDA、Fe^(2+)、ROS水平高于OGD/R+miR-22-3p组(t分别=3.78、3.87、3.05,P均<0.05)。结论miR-22-3p靶向调控PLAGL2表达抑制铁死亡,进而降低OGD/R诱导的心肌细胞损伤。
Objective To study the role of miR-22-3p in oxygen and glucose deprivation/reoxygenation(OGD/R)in⁃duced myocardial cell injury and the mechanism.Methods Rat myocardial cell line H9c2 was cultured upon OGD/R and transfetced with miR-22-3p mimic.Based on the presence or absence of hypoxia and transfection,cells were divided into normal oxygen group,OGD/R group,OGD/R+miRNA-NC group,and OGD/R+miR-22-3p mimic group.Cell viability and ferroptosis markers were detected by CCK-8 and ELISA methods.The expression of Gpx4 and PLAGL2 proteins were detected by western blot.Dual luciferase reporting assay verified the targeting relationship between miR-22-3p and PLA⁃GL2.Results After OGD/R treatment,the absorbance value of H9c2 cells on 4th day was lower than that of the normal oxygen group,the accumulation of ROS,MDA and Fe^(2+)was significantly higher than that of the normal oxygen group,the level of miR-22-3p was significantly lower than that of the normal oxygen group(t=3.78,4.24,2.54,4.47,3.53,P<0.05).After transfected with miR-22-3p mimic and subjected to OGD/R in H9c2 cells,the absorbance was higher than that of miR-NC+OGD/R group at third and fourth day(t=2.98,1.97,2.57,2.46,P<0.05).MDA,ROS and Fe^(2+)were sig⁃nificantly lower than those in OGD/R+miR-NC group(t=2.62,5.35,4.11,P<0.05),and Gpx4 expression level was high⁃er than that in OGD/R+miR-NC group.After Fer-1 and OGD/R treatment,the absorbance of H9c2 cells at third and fourth day was significantly higher than that of OGD/R group(t=3.12,3.36,P<0.05).The levels of MDA,ROS and Fe^(2+)were significantly lower than those of OGD/R group(t=3.03,5.33,2.48,P<0.05).The PLAGL2 protein expression in H9c2 cells transfected with miR-22-3p group was significantly lower than that of miR-NC group.The luciferase activity in PLAGL2-WT+miR-22-3p group was significantly lower than that of PLAGL2-MUT+miR-22-3p group(t=2.69,P<0.05).After co-transfected with miR-22-3p mimic and PLAGL2 overexpression plasmid,the Gpx4 expression in H9c2 cells was lower than that of OGD/R+miR-22-3p group,and the absorbance was significantly lower than that of OGD/R+miR-22-3p group(t=3.91,3.12,P<0.05).The levels of MDA,Fe^(2+)and ROS were higher than those of OGD/R+miR-22-3p group(t=3.78,3.87,3.05,P<0.05).Conclusion miR-22-3p inhibits ferroptosis and reduces OGD/R-induced myocardial cell injury via targeting PLAGL2.
作者
陈玥璇
汪潞
柳华锋
CHEN Yuexuan;WANG Lu;LIU Huafeng(Department of Emergency,Hangzhou Traditional Chinese Medicine Hospital,Hangzhou 310007,China)
出处
《全科医学临床与教育》
2023年第9期784-789,共6页
Clinical Education of General Practice
