首都医科大学学报 ›› 2014, Vol. 35 ›› Issue (3): 305-309.doi: 10.3969/j.issn.1006-7795.2014.03.009

• 脑血管病的基础研究 • 上一篇    下一篇

miRNA-99a对过氧化氢诱导neuro-2a细胞氧化损伤的影响

陶真, 王荣亮, 赵海苹, 罗玉敏   

  1. 首都医科大学宣武医院脑血管病研究室, 北京 100053
  • 收稿日期:2014-02-17 出版日期:2014-06-21 发布日期:2014-06-14
  • 通讯作者: 罗玉敏,E-mail:yumin111@ccmu.edu.cn E-mail:yumin111@ccmu.edu.cn
  • 基金资助:

    国家自然科学基金项目(81201028,81271461)。

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

Tao Zhen, Wang Rongliang, Zhao Haiping, Luo Yumin   

  1. Cerebrovascular Diseases Research Institute, Xuanwu Hospital, Capital Medical University, Beijing 100053, China
  • Received:2014-02-17 Online:2014-06-21 Published:2014-06-14
  • Supported by:

    This study was supported by National Natural Science Foundation of China (81201028, 81271461).

摘要:

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100 μmol/L刺激组,miR-99a预处理组(转染miRNA-99a mimics+过氧化氢100 μmol/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 25 000,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。

关键词: miR-99a, neuro-2a, 过氧化氢, 氧化损伤

Abstract:

Objective To investigate the effects of microRNA-99a(miR-99a) on neuro-2a cells against oxidative injury induced by hydrogen peroxide(H2O2). Methods Neuro-2a cells were divided into 3 groups by different treatments: control group, control+H2O2(100 μmol/L) group and miR-99a mimics+H2O2 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+H2O2 group and miR-99a+H2O2 group, decreased significantly to 85% and 89% respectively upon hydrogen peroxide stimulation(P<0.05). However, miR-99a+H2O2 group appeared to have less reduction of 11% than control+H2O2 group(P<0.05). Further studies showed that enzyme activities of T-SOD and Mn-SOD were depressed in control+H2O2 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+H2O2 group(P<0.05). Besides, NADH content of neuro-2a in control+H2O2 group was reduced compared with control group(P<0.05), while in miR-99a+H2O2 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+H2O2 group had a tendency of more increase in protein expression of SNAP25, compared to control+H2O2 group. Conclusion miR-99a can effectively protect neuro-2a cells from oxidative injury induced by hydrogen peroxide.

Key words: miR-99a, neuro-2a, H2O2, oxidative injury

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