miRNA-99a对过氧化氢诱导neuro-2a细胞氧化损伤的影响

doi:10.3969/j.issn.1006-7795.2014.03.009

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

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

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

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/Abstract

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

中图分类号:

R743.9

陶真, 王荣亮, 赵海苹, 罗玉敏. miRNA-99a对过氧化氢诱导neuro-2a细胞氧化损伤的影响[J]. 首都医科大学学报, 2014, 35(3): 305-309.

Tao Zhen, Wang Rongliang, Zhao Haiping, Luo Yumin. Effects of microRNA-99a on Neuro-2a cells against oxidative injury induced by hydrogen peroxide[J]. Journal of Capital Medical University, 2014, 35(3): 305-309.

参考文献

[1] van Spronsen M, van Battum E Y, Kuijpers M, et al. Developmental and activiry-dependent miRNA expression profiling in primary hippocampal neuron cultures[J]. PLoS One, 2013, 8(10):e74907.
[2] Wang L, Chang L, Li Z, et al. MiR-99a and -99b inhibit cervical cancer cell proliferation and invasion by targeting mTOR signaling pathway[J]. Med Oncol, 2014, 31(5):934.
[3] Coppola A, Romito A, Borel C, et al. Cardiomyogenesis is controlled by the miR-99a/let-7c cluster and epigenetic modifications[J]. Stem Cell Res, 2014, 12(2):323-337.
[4] Qin S, Zhang C. MicroRNAs in vascular disease[J]. J Cardiovasc Pharmacol, 2011, 57(1):8-12.
[5] Zhao H, Wang J, Gao L, et al. MiRNA-424 protects against permanent focal cerebral ischemia injury in mice involving suppressing microglia activation[J]. Stroke, 2013, 44(6):1706-1713.
[6] Liu X, Li F, Zhao S, et al. MicroRNA-124-mediated regulation of inhibitory member of apoptosis-stimulating protein of p53 family in experimental stroke[J]. Stroke, 2013, 44(7):1973-1980.
[7] Zhu F, Liu J L, Li J P, et al. MicroRNA-124(miR-124) regulates Ku70 expression and is correlated with neuronal death induced by ischemia/reperfusion[J]. J Mol Neurosci, 2014, 52(1):148-155.
[8] Chen Z, Jin Y, Yu D, et al. Down-regulation of the microRNA-99 family members in head and neck squamous cell carcinoma[J]. Oral Oncol, 2012, 48(8):686-691.
[9] Cui L, Zhou H, Zhao H, et al. MicroRNA-99a induces G1-phase cell cycle arrest and suppresses tumorigenicity in renal cell carcinoma[J]. BMC Cancer, 2012, 12:546.
[10] Sun D, Lee Y S, Malhotra A, et al. MiR-99 family of microRNAs suppresses the expression of prostate-specific antigen and prostate cancer cell proliferation[J]. Cancer Res, 2011, 71(4):1313-1324.
[11] 张连富, 徐善水, 方兴根.microRNAs在动脉瘤形成中作用的研究进展[J].中国脑血管病杂志, 2013, 10(4):55-62.
[12] Lerman G, Avivi C, Mardoukh C, et al. MiRNA expression in psoriatic skin: reciprocal regulation of has-miR-99a and IGF-1R[J]. PLoS One, 2011, 6(6):e20916.
[13] Turcatel G, Rubin N, EI-Hashash A, et al. MIR-99a and MIR-99b modulate TGF-β induced epithelial to mesenchymal plasticity in normal murine mammary gland cells[J]. PLoS One, 2012, 7(1):e31032.
[14] Yu Y, Du J R, Wang C Y, et al. Protection against hydrogen peroxide induced injury by Z-ligustilide in PC12 cells[J]. Exp Brain Res, 2008, 184(3):307-312.
[15] Lu X, Xu H, Sun B, et al. Enhanced neuroprotective effects of resveratrol delivered by nanoparticles on hydrogen peroxide-induced oxidative stress in rat cortical cell culture[J]. Mol Pharm, 2013, 10(5):2045-2053.
[16] Liu Q, Kou J P, Yu B Y. Ginsenoside Rg1 protects against hydrogen peroxide-induced cell death in PC12 cells via inhibiting NF-κB activation[J]. Neurochem Int, 2011, 58(1):119-125.
[17] Tretter L, Adam-Vizi V. Inhibition of Krebs cycle enzymes by hydrogen peroxide:a key role of[alpha]-ketoglutarate dehydrogenase in limiting NADH production under oxidative stress[J]. J neurosci, 2000, 20(24):8972-8979.
[18] Cao W, Razanau A, Feng D, et al. Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation[J]. Nucleic Acids Res, 2012, 40(16):8059-8071.

miRNA-99a对过氧化氢诱导neuro-2a细胞氧化损伤的影响

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

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