miRNA-99a 对过氧化氢诱导 neuro-2a 细胞氧化损伤的影响

目的：研究 microRNA-99a（miR-99a）对过氧化氢所诱导神经细胞 neuro-2a 氧化损伤的影响。方法常规培养 neuro-2a细胞并分为3组：正常对照组,过氧化氢100μmol/ L 刺激组,miR-99a 预处理组（转染 miRNA-99a mimics＋过氧化氢100μmol/ L刺激）,用 CCK-8试剂盒检测细胞存活率,生化试剂盒检测还原型烟酰胺腺嘌呤二核苷酸（ nicotinamide adenine dinucleotide, NADH）含量和总超氧化物歧化酶（total superoxide dismutase,T-SOD）、锰超氧化物歧化酶（manganese superoxide dismutase,Mn-SOD）活性,Western blotting 检测突触小体相关蛋白（synaptosoma associated protein of molecular mass 25000,SNAP25）及 Mn-SOD、细胞外超氧化物歧化酶（extracellular SOD,EC-SOD）的蛋白表达水平。结果与正常对照组相比,过氧化氢刺激的2组细胞存活率明显下降,分别为85%和89%（P〈0.05）,但 miR-99a 预处理组的细胞存活率下降程度较小仅为11%（P〈0.05）。进一步研究发现,过氧化氢刺激引起细胞 T-SOD、Mn-SOD 活性降低（P〈0.05）,转染 miR-99a mimics 不但能增强 T-SOD 和 Mn-SOD 的活性,还能促进 Mn-SOD 和 EC-SOD 的表达（P〈0.05）。过氧化氢导致细胞中 NADH 含量降低（P〈0.05）,miR-99a 能够增加 NADH 含量甚至高于正常细胞水平（P〈0.05）。 miR-99a 还有增加 neuro-2a 细胞表达 SNAP25的趋势。结论 miR-99a 对过氧化氢诱导neuro-2a 细胞引起的氧化损伤具有保护作用。

Effects of microRNA-99a on Neuro-2a cells against oxidative injury induced by hydrogen peroxide

Objective To investigate the effects of microRNA-99a（miR-99a） on neuro-2a cells against oxidative injury induced by hydrogen peroxide（H2 O2 ）. Methods Neuro-2a cells were divided into 3 groups by different treatments： control group, control＋H2 O2 （100 μmol/ L） group and miR-99a mimics＋H2 O2 group. Cell viability was measured by cell counting kit-8. Total superoxide dismutase （T-SOD） activity, manganese superoxide dismutase （ Mn-SOD） activity, and content of reduced nicotinamide adenine dinucleotide （NADH） were assayed by a variety of biochemical kits according to the instructions respectively. Protein expression levels of synaptosoma associated protein of molecular mass 25 000（SNAP25）, Mn-SOD and extracellular SOD（EC-SOD） were assessed by Western blotting. Results Compared with control group, cell viability of neuro-2a in the control ＋ H2 O2 group and miR-99a ＋ H2 O2 group, decreased significantly to 85% and 89% respectively upon hydrogen peroxide stimulation（P〈0. 05）. However, miR-99a＋H2 O2 group appeared to have less reduction of 11% than control＋H2 O2 group（P〈0. 05）. Further studies showed that enzyme activities of T-SOD and Mn-SOD were depressed in control＋H2 O2 group in comparison with control group（ P〈0. 05）. Whereas, with pretreatment of miR-99a mimics transfection, T-SOD and Mn-SOD activities were enhanced to a significantly higher level, relative to the normal level of control group（P〈0. 05）, as well as greatly increased protein expression of Mn-SOD and EC-SOD, in contrast to control group and control＋H2 O2 group（P〈0. 05）. Besides, NADH content of neuro-2a in control＋H2 O2 group was reduced compared with control group（P〈0. 05）, while in miR-99a＋H2 O2 group, NADH content exceeded the normal level of control group（ P〈0. 05）. It was also found that protein expression of SNAP25 increased in the two groups of hydrogen peroxide stimulation. And miR-99a＋H2 O2 group had a tendency of more increase in protein expression of SNAP25, compared to control ＋ H2 O2 group. Conclusion miR-99a can effectively protect neuro-2a cells from oxidative injury induced by hydrogen peroxide.