神经源性膀胱miRNA-mRNA调控网络的筛选与验证

Screening and validation of neurogenic bladder miRNA-mRNA regulatory network

背景:miRNA-mRNA调控网络在各种生物过程中发挥重要作用,但其在脊髓损伤后神经源性膀胱中的具体作用机制尚不清楚。目的:基于生物信息学挖掘神经源性膀胱的相关靶点,并构建miRNA-mRNA调控网络,加以动物实验验证。方法:从GEO数据库筛选神经源性膀胱的差异基因,构建miRNA-mRNA调控网络。利用David数据库对差异基因进行GO和KEGG分析,利用STRING数据库进行PPI分析,并通过Cytoscape和ROC曲线筛选关键基因,最终通过神经源性膀胱大鼠模型对关键基因进行RT-PCR验证。结果与结论:①共筛选出188个神经源性膀胱的差异基因,GO及KEGG分析发现其主要在炎症反应、Cytokine-cytokine受体相互作用、趋化因子信号通路等方面显著富集;通过Cytoscape及ROC曲线结果筛选出miR-147-脑源性神经营养因子和miR-362-5p-Scn9a 2个关键调控网络;②RT-PCR检测结果显示,与对照组比较,神经源性膀胱模型大鼠中miR-147、miR-362-5p、脑源性神经营养因子表达量上调,Scn9a基因下调;③结果提示,miR-147-脑源性神经营养因子、miR-362-5p-Scn9a作为炎症、氧化应激等病理生理过程的重要调控因子,有望成为诊断和治疗神经源性膀胱的新靶点。

BACKGROUND:The miRNA-mRNA regulatory network plays an important role in various biological processes,but its specific mechanism in neurogenic bladder after spinal cord injury remains unclear.OBJECTIVE:To mine the related targets of neurogenic bladder based on bioinformatics and to construct the miRNA-mRNA regulatory network,verified by animal experiments.METHODS:The differential genes of neurogenic bladder were screened from GEO database to construct the miRNA-mRNA regulatory network.The differential genes were analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses using the David database.Protein-protein interaction analysis was performed using the STRING database.Key genes were identified by Cytoscape and ROC curves.Finally the key genes were validated by RT-PCR in a rat model of neurogenic bladder.RESULTS AND CONCLUSION:A total of 188 differential genes related to neurogenic bladder were identified.Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that they were enriched mainly in inflammatory response,cytokine-cytokine receptor interaction,and chemokine signaling pathway.Two key regulatory networks,miR-147-brain-derived neurotrophic factor and miR-362-5p-Scn9a,were identified based on the Cytoscape and ROC curve results.RT-PCR results showed that miR-147,miR-362-5p,and brain-derived neurotrophic factor were highly expressed in the neurogenic bladder rats,while Scn9a was lowly expressed compared with the control rats.Overall,these findings indicate that miR-147-brain-derived neurotrophic factor and miR-362-5p-Scn9a actas important regulators of pathophysiological processes such as inflammation and oxidative stress,which are expected to become new targets for the diagnosis and treatment of neurogenic bladder.