MicroRNA-98对糖氧剥夺诱导的SH-SY5Y细胞损伤的影响

The effect of microRNA-98 on SH-SY5Y cell damage induced by oxygen-glucose deprivation

摘要:

目的 探讨microRNA-98（miR-98）在人神经母细胞瘤细胞SH-SY5Y中的表达及与缺血性脑卒中细胞凋亡和炎症损伤的关系。方法 采用氧糖剥夺/再灌注（OGD/R）诱导SH-SY5Y细胞构建体外缺血性脑卒中细胞模型。细胞分为对照组（未经任何处理）和OGD/R组，采用MTT法检测细胞活力，采用实时荧光定量聚合酶链反应（qRT-PCR）检测miR-98、Rho相关卷曲螺旋激酶2（ROCK2）表达；通过生物信息学的方法，寻找与ROCK2结合特异性好、稳定性强的miRNAs，采用双萤光素酶报告基因检测萤光素酶活性。Western blotting检测ROCK2蛋白相对表达量。将miR-98模拟物、miR-98抑制剂、pcDNA3.1-ROCK2和相应的阴性对照组转染到SH-SY5Y细胞中，并进行OGD/R处理。采用流式细胞术、TUNEL法检测细胞凋亡情况；采用吸光光度法检测凋亡相关蛋白胱天蛋白酶3（Caspase-3）和胱天蛋白酶9（Caspase-9）水平；qRT-PCR检测TNF-α和IL-6表达。结果 与对照组相比，OGD/R组细胞活力降低，miR-98相对表达量降低（P <0.05），ROCK2 mRNA相对表达量升高（P <0.05）。联合生物信息学和双萤光素酶实验分析发现ROCK2是miR-98的靶点之一（P <0.05）。与对照组相比，miR-98模拟物转染组miR-98 mRNA相对表达量升高（P <0.05），而ROCK2蛋白相对表达量降低（P <0.05）；miR-98抑制剂转染组miR-98 mRNA相对表达量降低，而ROCK2蛋白相对表达量升高（P <0.05）。与对照组相比，OGD/R组细胞凋亡率、Caspase-3和Caspase-9水平升高（P <0.05）；与模拟物对照组相比，miR-98模拟物组细胞凋亡率、Caspase-3和Caspase-9水平降低（P <0.05）；与miR-98模拟物组相比，miR-98模拟物组+pcDNA3.1-ROCK2组的细胞凋亡率、Caspase-3和Caspase-9水平升高（P <0.05）。与对照组相比，OGD/R组TNF-α和IL-6 mRNA相对表达量升高（P <0.05）；与模拟物对照组相比，miR-98模拟物组TNF-α和IL-6 mRNA相对表达量降低（P <0.05）；与miR-98模拟物组相比，miR-98模拟物组+pcDNA3.1-ROCK2组的TNF-α和IL-6 mRNA相对表达量升高（P <0.05）。结论 miR-98通过靶向抑制ROCK2的表达，抑制神经母细胞瘤SH-SY5Y细胞的凋亡和炎症损伤，在缺血性脑卒中神经元损伤中发挥保护作用。

Abstract:

Objective To explore the expression of microRNA-98 (miR-98) in human neuroblastoma cell line SH-SY5Y and its relationship with cell apoptosis and inflammatory injury in ischemic stroke.Methods Oxygen-glucose deprivation/reperfusion (OGD/R) was used to induce SH-SY5Y cells to establish an in vitro cell model of ischemic stroke. The cells were divided into control group (without any treatment) and OGD/R group. Cell viability was detected by MTT assay. The expression levels of miR-98 and Rho-associated coiled-coil kinase 2 (ROCK2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics methods were used to screen miRNAs that specifically bind to ROCK2 with high stability, and dual-luciferase reporter gene assay was performed to detect luciferase activity. The relative expression level of ROCK2 protein was detected by Western blotting. miR-98 mimics, miR-98 inhibitors, pcDNA3.1-ROCK2 and their corresponding negative controls were transfected into SH-SY5Y cells, followed by OGD/R treatment. Flow cytometry and TUNEL assay were used to detect cell apoptosis. Absorptiometry was used to determine the levels of apoptotic-related proteins Caspase-3 and Caspase-9. The mRNA expression levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected by qRT-PCR.Results Compared with the control group, the OGD/R group showed decreased cell viability, reduced relative expression level of miR-98 (P < 0.05), and increased relative expression level of ROCK2 mRNA (P < 0.05). Combined bioinformatics analysis and dual-luciferase reporter gene assay confirmed that ROCK2 is one of the target genes of miR-98 (P < 0.05). Compared with the control group, the miR-98 mimics transfection group had an increased relative expression level of miR-98 mRNA (P < 0.05) and a decreased relative expression level of ROCK2 protein (P < 0.05); the miR-98 inhibitor transfection group had a decreased relative expression level of miR-98 mRNA and an increased relative expression level of ROCK2 protein (P < 0.05). Compared with the control group, the OGD/R group had increased cell apoptosis rate and elevated levels of Caspase-3 and Caspase-9 (P < 0.05); compared with the mimic control group, the miR-98 mimics group had decreased cell apoptosis rate and reduced levels of Caspase-3 and Caspase-9 (P < 0.05); compared with the miR-98 mimics group, the miR-98 mimics + pcDNA3.1-ROCK2 group had increased cell apoptosis rate and elevated levels of Caspase-3 and Caspase-9 (P < 0.05). Compared with the control group, the OGD/R group had increased mRNA expression levels of TNF-α and IL-6 (P < 0.05); compared with the mimic control group, the miR-98 mimics group had decreased mRNA expression levels of TNF-α and IL-6 (P < 0.05); compared with the miR-98 mimics group, the miR-98 mimics + pcDNA3.1-ROCK2 group had increased mRNA expression levels of TNF-α and IL-6 (P < 0.05).Conclusion miR-98 inhibits apoptosis and inflammatory injury of human neuroblastoma SH-SY5Y cells by targeting and inhibiting the expression of ROCK2, thereby exerting a protective effect against neuronal injury in ischemic stroke.