MicroRNA-98对糖氧剥夺诱导的SH-SY5Y细胞损伤的影响OA

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.