LncRNA MIAT靶向miR-204对氧糖剥夺/复氧诱导PC12细胞损伤的保护作用

Protective effect of lncRNA MIAT-targeted miR-204 against OGD/R-induced PC12 cell injury

目的探究氧糖剥夺/复氧(OGD/R)诱导大鼠肾上腺髓质嗜铬瘤分化细胞株PC12细胞损伤的分子调控机制,以期为临床上靶向治疗缺血性卒中提供新的思路。方法该研究于2021年6—12月进行,购买PC12细胞后对其进行OGD/R诱导,在诱导后的细胞中分别转染长链非编码RNA(LncRNA)心肌梗死相关转录本(MIAT)过表达载体及微RNA-204模拟物(miR-204 mimic),以对应的载体阴性对照(pcDNA3.1-NC)或模拟物阴性对照(mimic-NC)作为阴性对照,以未转染PC12细胞作为空白对照。使用实时荧光定量聚合酶链式反应(qRT-PCR)检测LncRNA MIAT与miR-204的表达;CCK-8与流式细胞术分别检测细胞活力与凋亡;Elisa试剂盒检测炎性因子白细胞介素(IL)-6、IL-1β,抗炎性因子IL-10的表达。RNA下拉检测MIAT在miR-204上的富集;通过starbase预测LncRNA MIAT与miR-204的结合位点,随后采取双萤光素酶报告实验验证LncRNA MIAT与miR-204的靶向结合。结果与空白对照组[1.011±0.113,1.001±0.002,1.473±0.224,(8.16±0.84)%,(96.75±6.73)ng/L,(46.28±2.84)ng/L,(39.45±1.45)ng/L]相比,OGD/R组细胞中LncRNA MIAT表达显著降低(0.362±0.085),miR-204表达显著升高(2.234±0.306),细胞活力显著降低0.806±0.115,凋亡率显著增加[(28.25±4.13)%];炎性因子IL-6[(525.19±15.62)ng/L]、IL-1β[(292.54±19.54)ng/L]的表达显著增加,抗炎性因子IL-10的表达(14.33±2.36)ng/L显著降低(均P<0.01)。与阴性对照组相比[2.198±0.324,0.811±0.117,(8.16±0.84)%,(96.75±6.73)ng/L,(46.28±2.84)ng/L,(39.45±1.45)ng/L],MIAT过表达后OGD/R细胞中miR-204表达显著降低1.373±0.268,细胞活力显著升高1.137±0.116,凋亡率显著降低(28.25±4.13)%;炎性因子IL-6(525.19±15.62)ng/L、IL-1β(292.54±19.54)ng/L的表达显著降低,抗炎性因子IL-10(14.33±2.36)ng/L的表达显著升高(均P<0.01)。与MIAT过表达的OGD/R细胞相比,MIAT过表达载体和miR-204模型物共转染的OGD/R细胞中miR-204表达显著升高,细胞活力显著降低,凋亡率显著升高;炎性因子IL-6、IL-1β的表达显著升高,抗炎性因子IL-10的表达显著降低(均P<0.01)。结论在OGD/R诱导的PC12细胞中,低表达LncRNA MIAT促进miR-204表达上调,最终促进细胞损伤。

Objective To investigate the molecular regulatory mechanism of oxygen–glucose deprivation/reoxygenation(OGD/R)-in⁃duced PC12 cell injury in a rat adrenal medullary pheochromocytoma differentiated cell line,with the aim of providing new ideas for clinically targeted therapy of ischemic stroke.Methods The study was conducted from June to December 2021.PC12 cells were pur⁃chased and subjected to OGD/R induction,in which the induced cells were transfected with the long noncoding RNA(lncRNA)myocar⁃dial infarction-associated transcript(MIAT)overexpression vector and microRNA-204 mimic(miR-204 mimic),respectively,and a neg⁃ative control of the corresponding vector(pcDNA3.1-NC)or mimic negative control(mimic-NC)as a negative control and untransfected PC12 cells as a blank control.The expression of lncRNA MIAT and miR-204 was detected using real-time fluorescence quantitative polymerase chain reaction(qRT‒PCR);cell viability and apoptosis were detected by CCK-8 and flow cytometry,respectively;and the expression of the inflammatory factors interleukin(IL)-6 and IL-1,and the anti-inflammatory factor IL-10 was detected by enzyme�linked immunosorbent assay(ELISA).The enrichment of the LncRNA MIAT on miR-204 was detected by RNA pull-down.The binding sites of LncRNA MIAT to miR-204 were predicted by starBase,and subsequently a dual-luciferase reporter assay was taken to verify the targeted binding of lncRNA MIAT to miR-204.Results Compared with the blank control group[1.011±0.113,1.001±0.002,1.473±0.224,(8.16±0.84)%,(96.75±6.73)ng/L,(46.28±2.84)ng/L,and(39.45±1.45)ng/L],the expression of the LncRNA MIAT was significantly decreased(0.362±0.085),miR-204 expression was significantly increased(2.234±0.306),cell viability was significantly decreased(0.806±0.115),and the apoptosis rate was significantly increased(28.25±4.13)%in OGD/R group.The expression of the in⁃flammatory factors IL-6(525.19±15.62)ng/L and IL-1(292.54±19.54)ng/L was significantly increased,and the expression of anti-in⁃flammatory factor IL-10(14.33±2.36)ng/L was significantly decreased(P<0.01).Compared with the negative control group[2.198±0.324,0.811±0.117,(8.16±0.84)%,(96.75±6.73)ng/L,(46.28±2.84)ng/L,(39.45±1.45)ng/L],the expression of miR-204 was signifi⁃cantly reduced in OGD/R cells after MIAT overexpression(1.373±0.268),cell viability was significantly increased(1.137±0.116),and the apoptosis rate was significantly decreased(28.25±4.13)%.The expression of the inflammatory factors IL-6(525.19±15.62)and IL-1(292.54±19.54)was significantly decreased,and the expression of the anti-inflammatory factor IL-10(14.33±2.36)expression was sig⁃nificantly higher(P<0.01).Compared with MIAT-overexpressing OGD/R cells,miR-204 expression was significantly higher,cell via⁃bility was significantly lower,and apoptosis rate was significantly higher in OGD/R cells cotransfected with the-MIAT overexpressing vector and miR-204 model.The expression of the inflammatory factors IL-6 and IL-1 was significantly elevated,and the expression of the anti-inflammatory factor IL-10 was significantly reduced(P<0.01).Conclusion In OGD/R-induced PC12 cells,low expression of the lncRNA MIAT promotes the upregulation of miR-204 expression and ultimately cell injury.