miR-20a-3p靶向STAT3调控大鼠脊髓损伤轴突修复
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摘要
目的探索Wistar大鼠脊髓损伤后损伤局部微环境发生改变的分子生物学机制,寻找起关键调控作用的微RNA。方法 15只雌性Wistar大鼠随机分为对照组(n=3)和脊髓损伤组(n=12)。脊髓损伤组根据取材时间分为4 h、3d、7 d、14 d四个组,每组3只。使用Microarray 3.0芯片检测脊髓损伤大鼠损伤局部发生改变的微RNA,运用生物信息学方法论证发挥关键调控作用的微RNA,进行靶基因预测。运用逆转录实时定量聚合酶链反应(RT-qPCR)技术检测miR-20a-3p表达。采用Western blotting技术检测信号传导子和转录激活子(STAT)3表达量,并分析各组中靶蛋白与目标微RNA表达变化趋势相关性。在神经元中抑制关键候选微RNA,并使用免疫荧光观察靶蛋白表达与轴突生长的关系。结果脊髓损伤标本中miR-20a-3p特征性上调明显。生物信息分析结果显示,STAT3可为miR-20a-3p的靶基因,与其趋势相反。细胞实验结果显示,miR-20a-3p抑制组与对照组相比轴突延长。Western blotting结果显示,与对照组相比,miR-20a-3p抑制组STAT3蛋白表达显著上调。结论脊髓损伤后miR-20a-3p通过调节其序列互补靶基因STAT3表达量来影响神经元轴突的生长。miR-20a-3p上调导致STAT3下调,抑制miR-20a-3p,可促进神经元轴突的再生。
Objective To study the molecular biology mechanisms of Wistar rats after spinal cord injury, and find out key microRNAs.Methods A total of 15 Wistar rats were divided into control group(n = 3) and spinal cord injury group(n = 12). The latter group was divided into four hours, three days, seven days and 14 days subgroups, with three rats in each subgroup. Microarray 3.0 was used to investigate microRNA expression profiles of Wistar rats with spinal cord injury. Bioinformatics was used to predict microRNAs playing key regulatory roles, and to predict target genes. Reverse transcription real-time quantitative polymerase chain reaction(RT-qPCR) was applied to detect the expression of miR-20 a-3 p. Western blotting was employed to detect the signal transducer and activator of transcription(STAT) 3 level. The correlation between target protein and microRNA expression trend in each group was analyzed. The key microRNA was inhibited in the neurons. The relationship between target protein expression and axon growth was observed with immunofluorescence.Results In the rats with spinal cord injury, totally 658 microRNAs had changed at least once. In all the altered microRNAs, miR-20 a-3 p was upregulated obviously. It predicted that the target gene of miR-20 a-3 p was STAT3 via application of bioinformatics analysis. The expression trend of STAT 3 and miR-20 a-3 p in spinal cord was opposite.After the inhibition of miR-20 a-3 p, the expression of STAT3 in neurons was unregulated and axonal growth was extended.Conclusion The upregulation of miR-20 a-3 p leads to downregulation of STAT3, and miR-20 a-3 p is one of the key targets in the treatment of spinal cord injury.
Objective To study the molecular biology mechanisms of Wistar rats after spinal cord injury, and find out key microRNAs.Methods A total of 15 Wistar rats were divided into control group(n = 3) and spinal cord injury group(n = 12). The latter group was divided into four hours, three days, seven days and 14 days subgroups, with three rats in each subgroup. Microarray 3.0 was used to investigate microRNA expression profiles of Wistar rats with spinal cord injury. Bioinformatics was used to predict microRNAs playing key regulatory roles, and to predict target genes. Reverse transcription real-time quantitative polymerase chain reaction(RT-qPCR) was applied to detect the expression of miR-20 a-3 p. Western blotting was employed to detect the signal transducer and activator of transcription(STAT) 3 level. The correlation between target protein and microRNA expression trend in each group was analyzed. The key microRNA was inhibited in the neurons. The relationship between target protein expression and axon growth was observed with immunofluorescence.Results In the rats with spinal cord injury, totally 658 microRNAs had changed at least once. In all the altered microRNAs, miR-20 a-3 p was upregulated obviously. It predicted that the target gene of miR-20 a-3 p was STAT3 via application of bioinformatics analysis. The expression trend of STAT 3 and miR-20 a-3 p in spinal cord was opposite.After the inhibition of miR-20 a-3 p, the expression of STAT3 in neurons was unregulated and axonal growth was extended.Conclusion The upregulation of miR-20 a-3 p leads to downregulation of STAT3, and miR-20 a-3 p is one of the key targets in the treatment of spinal cord injury.
引文
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[3]王天仪,原文琦,刘勇,等.坐骨神经预损伤后miR-124-3p调节GAP-43表达并促进轴突生长的实验研究[J].中华骨科杂志,2015, 35(4):420-427.
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[6] Wang T, Yuan W, Liu Y, et al. miR-142-3p is a potential thera‐peutic target for sensory function recovery of spinal cord injury[J]. Med Sci Monit, 2015, 21:2553-2556.
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[11] Hauk T G, Leibinger M, Müller A, et al. Stimulation of axon regeneration in the mature optic nerve by intravitreal applica‐tion of the toll-like receptor 2 agonist Pam3Cys[J]. Invest Oph‐thalmol Vis Sci, 2010, 51(1):459.
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[13] Su L N, Song X Q, Xue Z X, et al. Network analysis of mi‐croRNAs, transcription factors, and target genes involved in ax‐on regeneration[J]. J Zhejiang Univ Sci B, 2018, 19(4):293-304.
[14] Gaudet A D, Mandrekar-Colucci S, Hall J C, et al. miR-155deletion in mice overcomes neuron-intrinsic and neuron-extrin‐sic barriers to spinal cord repair[J]. J Neurosci, 2016, 36(32):8516-8532.
[15]王天仪,刘勇,原文琦,等.微小RNA-199a-5p下调后通过cjun氨基端激酶通路促进脊髓后索损伤修复的研究[J].中华实验外科杂志, 2015, 32(7):1498-1502.
[16]王天仪,刘勇,原文琦,等.坐骨神经预损伤下调脊髓后索损伤大鼠背根神经节miR-182进而促进CREB蛋白表达的实验研究[J].中华神经医学杂志, 2015, 14(1):14-21.
[17] Hao J, Li B, Duan H Q, et al. Mechanisms underlying the promotion of functional recovery by deferoxamine after spinal cord injury in rats[J]. Neural Regen Res, 2017, 12(6):959-968.
[18] Duan H Q, Wu Q L, Yao X, et al. Nafamostatmesilate attenu‐ates inflammation and apoptosis and promotes locomotor recov‐ery after spinal cord injury[J]. CNS Neurosci Ther, 2018, 24(5):429-438.
[19] Charron F. Axon guidance:gained in translation[J]. Neuron,2018, 99(1):1-2.
[20] Ko H R, Kwon I S, Hwang I, et al. Akt1-Inhibitor of DNA binding 2 is essential for growth cone formation and axon growth and promotes central nervous system axon regeneration[J]. Elife, 2016, pii:e20799.
[21] Neumann S, Woolf C J. Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord inju‐ry[J]. Neuron, 1999, 23(1):83-91.
[22]王志杰,郭晓玲,刘海英,等.儿童急性横贯性脊髓炎患儿脑脊液中miR-182-5p上调促进CREB蛋白表达的实验研究[J].中国实用医药, 2017, 12(16):67-68.
[23] Epstein Y, Perry N, Volin M, et al. miR-9a modulates mainte‐nance and ageing of Drosophila germline stem cells by limiting N-cadherin expression[J]. Nat Commun, 2017, 8(1):600.
[24]张衍军,冯世庆,王奇,等.大鼠脊髓损伤后趋化因子受体4时空分布与意义[J].中华实验外科杂志, 2013, 30(7):1406-1408.
[25] Janssen H L, Reesink H W, Lawitz E J, et al. Treatment of HCV infection by targeting microRNA[J]. N Engl J Med,2013, 368(18):1685-1694.
[2] Ding Y, Song Z, Liu J. Aberrant LncRNA expression profile in a contusion spinal cord injury mouse model[J]. Biomed Res Int, 2016, 2016:9249401.
[3]王天仪,原文琦,刘勇,等.坐骨神经预损伤后miR-124-3p调节GAP-43表达并促进轴突生长的实验研究[J].中华骨科杂志,2015, 35(4):420-427.
[4] Wang T, Li B, Yuan X, et al. MiR-20a plays a key regulatory role in the repair of spinal cord dorsal column lesion via PDZRhoGEF/RhoA/GAP43 axis in rat[J]. Cell Mol Neurobiol,2019, 39(1):87-98.
[5] Wang Z, Yuan W, Li B, et al. PEITC promotes neurite growth in primary sensory neurons via the miR-17-5p/STAT3/GAP-43axis[J]. J Drug Target, 2019, 27(1):82-93.
[6] Wang T, Yuan W, Liu Y, et al. miR-142-3p is a potential thera‐peutic target for sensory function recovery of spinal cord injury[J]. Med Sci Monit, 2015, 21:2553-2556.
[7]王天仪,原文琦,刘勇,等.坐骨神经预损伤后背根神经节中miRNomes改变对大鼠脊髓后索损伤修复的影响[J].中国脊柱脊髓杂志, 2014, 24(12):1090-1098.
[8] Wang T, Liu Y, Yuan W, et al. Identification of microRNAome in rat bladder reveals miR-1949 as a potential inducer of blad‐der cancer following spinal cord injury[J]. Mol Med Rep,2015, 12(2):2849-2857.
[9] Nagata K, Hama I, Kiryu-Seo S, et al. microRNA-124 is down regulated in nerve-injured motor neurons and it potentially tar‐gets mRNAs for KLF6 and STAT3[J]. Neuroscience, 2014,256:426-432.
[10] Kumar R, Sahu S K, Kumar M, et al. MicroRNA 17-5p regu‐lates autophagy in Mycobacterium tuberculosis-infected macro‐phages by targeting Mcl-1 and STAT3[J]. Cell Microbiol,2016, 18(5):679-691.
[11] Hauk T G, Leibinger M, Müller A, et al. Stimulation of axon regeneration in the mature optic nerve by intravitreal applica‐tion of the toll-like receptor 2 agonist Pam3Cys[J]. Invest Oph‐thalmol Vis Sci, 2010, 51(1):459.
[12]王天仪,原文琦,刘勇,等.白细胞介素1在脊髓损伤后局部微环境改变中的作用[J].中国医药, 2015, 10(7):1083-1085.
[13] Su L N, Song X Q, Xue Z X, et al. Network analysis of mi‐croRNAs, transcription factors, and target genes involved in ax‐on regeneration[J]. J Zhejiang Univ Sci B, 2018, 19(4):293-304.
[14] Gaudet A D, Mandrekar-Colucci S, Hall J C, et al. miR-155deletion in mice overcomes neuron-intrinsic and neuron-extrin‐sic barriers to spinal cord repair[J]. J Neurosci, 2016, 36(32):8516-8532.
[15]王天仪,刘勇,原文琦,等.微小RNA-199a-5p下调后通过cjun氨基端激酶通路促进脊髓后索损伤修复的研究[J].中华实验外科杂志, 2015, 32(7):1498-1502.
[16]王天仪,刘勇,原文琦,等.坐骨神经预损伤下调脊髓后索损伤大鼠背根神经节miR-182进而促进CREB蛋白表达的实验研究[J].中华神经医学杂志, 2015, 14(1):14-21.
[17] Hao J, Li B, Duan H Q, et al. Mechanisms underlying the promotion of functional recovery by deferoxamine after spinal cord injury in rats[J]. Neural Regen Res, 2017, 12(6):959-968.
[18] Duan H Q, Wu Q L, Yao X, et al. Nafamostatmesilate attenu‐ates inflammation and apoptosis and promotes locomotor recov‐ery after spinal cord injury[J]. CNS Neurosci Ther, 2018, 24(5):429-438.
[19] Charron F. Axon guidance:gained in translation[J]. Neuron,2018, 99(1):1-2.
[20] Ko H R, Kwon I S, Hwang I, et al. Akt1-Inhibitor of DNA binding 2 is essential for growth cone formation and axon growth and promotes central nervous system axon regeneration[J]. Elife, 2016, pii:e20799.
[21] Neumann S, Woolf C J. Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord inju‐ry[J]. Neuron, 1999, 23(1):83-91.
[22]王志杰,郭晓玲,刘海英,等.儿童急性横贯性脊髓炎患儿脑脊液中miR-182-5p上调促进CREB蛋白表达的实验研究[J].中国实用医药, 2017, 12(16):67-68.
[23] Epstein Y, Perry N, Volin M, et al. miR-9a modulates mainte‐nance and ageing of Drosophila germline stem cells by limiting N-cadherin expression[J]. Nat Commun, 2017, 8(1):600.
[24]张衍军,冯世庆,王奇,等.大鼠脊髓损伤后趋化因子受体4时空分布与意义[J].中华实验外科杂志, 2013, 30(7):1406-1408.
[25] Janssen H L, Reesink H W, Lawitz E J, et al. Treatment of HCV infection by targeting microRNA[J]. N Engl J Med,2013, 368(18):1685-1694.