儿童急性横贯性脊髓炎的潜在治疗靶点实验研究

An Experimental Study on Potential Therapeutic Target for Acute Transverse Myelitis in Children

目的探索在儿童急性横贯性脊髓炎(ATM)发生过程中起到关键调控作用的microRNA,寻找潜在的治疗靶点。方法应用microarray 4.0芯片技术对儿童ATM脑脊液中发生改变的microRNA进行检测。使用生物信息学方法来验证发挥重要调控作用的关键microRNA,并预测其靶基因;分析miRNA表达谱数据,检测miRNA表达情况。用ELISA与Western blot技术检测关键miRNA靶基因和蛋白表达量。在背根神经节神经元中抑制或过表达关键候选microRNA,进行体外功能验证。结果ATM患者脑脊液样本中miR-133b显著下调(P<0.05)。生物信息学分析证明miR-133b的靶基因是基质金属蛋白酶-9(MMP-9)。miR-133b过表达组背根神经节神经元轴突明显延长(P<0.05)。miR-133b抑制组神经元轴突生长明显弱于空白组(P<0.05)。miR-133b过表达组MMP-9蛋白的表达低于空白组(P<0.05)。miR-133b抑制组MMP-9蛋白的表达明显高于空白组(P<0.05)。结论儿童ATM脑脊液中miR-133b的下调导致了MMP-9蛋白的上调,进而抑制了脊髓病变区域局部轴突的再生,可以证明儿童ATM的潜在治疗靶点之一是miR-133b。

Objective To explore the microRNAs that played a key regulatory role in the occurrence of acute transverse myelitis(ATM)in children so as to look for potential therapeutic targets.Methods The altered microRNAs in cerebrospinal fluid of children with ATM were detected by microarray 4.0 chip technique.Bioinformatics method was used to verify the key microRNA that played a key regulatory role and predict its target gene.The miRNA expression profile data was analyzed,and miRNA expression was detected.ELISA and Western blot techniques were used to detect expressions of key miRNA target genes and proteins.The key candidate microRNAs were inhibited or overexpressed in dorsal root ganglion neurons for in vitro functional verification.Results The miR-133 b expressions were significantly down-regulated in cerebrospinal fluids of children with ATM(P<0.05).The target gene of miR-133 b was matrix metalloproteinases-9(MMP-9)according to bioinformatics analysis.The axons of dorsal root ganglion neurons in miR-133 b overexpression group were significantly elongated(P<0.05).The neuron axon growth in miR-133 b inhibition group was significantly weaker than that in blank control group(P<0.05).The expression of MMP-9 protein in miR-133 b overexpression group was significantly lower than that in blank control group(P<0.05).The expression of MMP-9 protein in miR-133 b inhibition group was significantly higher than that in blank control group(P<0.05).Conclusion The down-regulation of miR-133 b leads to up-regulation of MMP-9 in children with ATM,which in turn inhibits the regeneration of local axons in lesion area of the spinal cord.It may be proved that miR-133 b is one of the potential therapeutic targets in treatment of children with ATM.