Liu Wan-Gen, Han Li-Li, Xiang Rong
Department of Neurology, Cangzhou Central Hospital, Cangzhou, Hebei, China.
J Cell Physiol. 2019 Sep;234(9):16226-16237. doi: 10.1002/jcp.28286. Epub 2019 Feb 14.
To study the role and mechanism of microRNA 19b (miR-19b) in hypoxia/reoxygenation (H/R)-induced injury by targeting PTEN.
PC12 and BV2 cells induced by H/R were treated with miR-19b mimics/inhibitors or small interfering PTEN (si-PTEN), respectively. Lactate dehydrogenase (LDH) level, malondialdehyde (MDA), and superoxide dismutase (SOD) content was detected. Besides, cell viability and apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Hoechst33342 staining, and flow cytometry, whereas mitochondrial membrane potential (MMP) tested by JC-1 assay, and reactive oxygen species (ROS) evaluated by the dichloro-dihydro-fluorescein diacetate assay. The ischemia/reperfusion (I/R) rats model was used to investigate the effects of miR-19b in vivo test. The infarct area and apoptosis rates in brain tissues were detected by 2,3,5-triphenyltetrazolium chloride and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining, respectively. miR-19b and PTEN/PI3K/Akt pathway-related proteins were detected by quantitative reverse-transcription polymerase chain reaction and western blot analysis.
miR-19b mimics could reduce LDH, MDA, and ROS levels and decline cell apoptosis, but enhance the viability, MMP, and SOD activity with decreased PTEN and cleaved caspase, as well as increased p-Akt/Akt and Bcl-2/Bax ratios in H/R-induced PC12 and BV2 cells. However, miR-19b inhibitors led to completely opposite results to aggravate H/R-induced cell injury. Meanwhile, si-PTEN could reverse the effect of miR-19b inhibitors on H/R-induced injury. Moreover, treatment with miR-19b agomir after I/R in vivo sufficiently decreased infarct area and reduced apoptosis rates by targeting PTEN through the regulation of the PI3K/Akt pathway.
miR-19b could inhibit oxidative stress, enhance cell MMP, promote cell survival, and inhibit cell apoptosis by targeting PTEN via the regulation of the PI3K/Akt pathway, thus playing the neuronal protective effects.
通过靶向PTEN研究微小RNA 19b（miR-19b）在缺氧/复氧（H/R）诱导损伤中的作用及机制。
分别用miR-19b模拟物/抑制剂或小干扰PTEN（si-PTEN）处理H/R诱导的PC12和BV2细胞。检测乳酸脱氢酶（LDH）水平、丙二醛（MDA）和超氧化物歧化酶（SOD）含量。此外，通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐、Hoechst33342染色和流式细胞术测定细胞活力和凋亡，而通过JC-1检测法检测线粒体膜电位（MMP），通过二氯二氢荧光素二乙酸酯检测法评估活性氧（ROS）。采用缺血/再灌注（I/R）大鼠模型研究miR-19b在体内试验中的作用。分别通过2,3,5-三苯基氯化四氮唑和末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记染色检测脑组织中的梗死面积和凋亡率。通过定量逆转录聚合酶链反应和蛋白质免疫印迹分析检测miR-19b和PTEN/PI3K/Akt通路相关蛋白。
miR-19b模拟物可降低LDH、MDA和ROS水平，减少细胞凋亡，但增强细胞活力、MMP和SOD活性，同时降低PTEN和裂解的半胱天冬酶水平，并增加H/R诱导的PC12和BV2细胞中p-Akt/Akt和Bcl-2/Bax比值。然而，miR-19b抑制剂导致完全相反的结果，加重H/R诱导的细胞损伤。同时，si-PTEN可逆转miR-19b抑制剂对H/R诱导损伤的作用。此外，体内I/R后用miR-19b激动剂处理可通过调节PI3K/Akt通路靶向PTEN，充分减小梗死面积并降低凋亡率。
miR-19b可通过调节PI3K/Akt通路靶向PTEN，抑制氧化应激，增强细胞MMP，促进细胞存活并抑制细胞凋亡，从而发挥神经保护作用。
