转录组测序结合TMT蛋白质组学技术对脊髓损伤小鼠关键基因预测及相关病理机制研究

Prediction of key genes and study of related pathological mechanisms in mice with spinal cord injury by RNA sequencing combined with TMT proteomics technology

摘要:

目的 通过转录组测序（RNA sequencing，RNA-Seq）和TMT蛋白质组学技术，筛选差异表达的基因及蛋白，探讨脊髓损伤后复杂的病理机制。方法 50只雌性C57BL/6小鼠随机分为假手术组、模型组，每组25只。采用钳夹法在腰1处制备小鼠脊髓损伤模型，14 d后进行取材。采用后肢运动功能评分（basso mouse scale，BMS）评估小鼠后肢运动功能变化；HE染色法观察脊髓损伤区病理形态学变化；RNA-Seq技术筛选差异基因；TMT蛋白组学分析筛选差异蛋白；结合2种测序技术筛选变化趋势吻合的mRNA和蛋白并进行生物信息学分析。结果 与假手术组相比，模型组中BMS评分明显降低（P<0.05）；HE染色显示脊髓损伤区域结构疏松紊乱，出现空洞，细胞核固缩，炎性浸润严重，神经元坏死；RNA-Seq共筛选出565个差异mRNA，其中545个上调，20个下调，TMT蛋白组学共筛选出339个差异蛋白，其中278个上调，61个下调；2种测序的聚类热图显示2组样本的表达模式差异大；韦恩图分析获得83个趋势上调的mRNA或蛋白；蛋白互作（protein-protein interaction，PPI）网络分析获得11个核心靶点；GO富集分析显示分子功能或生物过程主要在免疫应答、溶酶体途径、细菌反应、液泡裂解等方面；KEGG富集通路主要在结核病变、溶酶体、吞噬小体途径等通路。结论 本研究筛选出的11个mRNA或蛋白可能是调控脊髓损伤病理过程的核心靶点，病理机制可能与免疫应答途径、溶酶体和吞噬小体等通路密切相关。

Abstract:

Objective To screen for differentially expressed genes and proteins by RNA sequencing（RNA-Seq） and tandem mass tag（TMT） proteomics analysis，and to explore the complex pathological mechanism of spinal cord injury（SCI）.Methods Fifty female C57BL/6 mice were equally randomized into two groups： sham group and model group. The SCI model was established by compressing the 1st lumbar vertebra with an aneurysm clip，and spinal cord tissues were harvested 14 days later. The hindlimb locomotor function was assessed by basso mouse scale（BMS）； pathomorphological changes of the injured area of the spinal cord were determined by hematoxylin-eosin（HE） staining； RNA-Seq was used to screen for differentially expressed genes，and TMT proteomics analysis was used to screen for differentially expressed proteins. The two sequencing techniques were combined to screen for the messenger RNAs（mRNAs） and proteins with consistent change trends and perform bioinformatics analysis.Results Compared with the sham group，the model group had significantly decreased BMS scores（P<0.05）. HE staining showed loose and disordered structure，cavitation，karyopyknosis，serious inflammatory infiltration，and neuronal necrosis in the injured area of the spinal cord. A total of 565 differentially expressed mRNAs（including 545 up-regulated mRNAs and 20 down-regulated mRNAs） were screened out by RNA-Seq，and 339 differentially expressed proteins（including 278 up-regulated proteins and 61 down-regulated proteins） were screened out by TMT proteomics analysis. The cluster heat maps of the two sequencing methods showed that the expression patterns of the two groups of samples were very different. A total of 83 up-regulated mRNAs or proteins were obtained by the Venn diagram. Eleven core targets were obtained by the protein-protein interaction network analysis. Gene ontology enrichment analysis showed that molecular functions or biological processes were mainly found in immune response，lysosome pathway，bacterial reaction，and vacuole lysis. Kyoto encyclopedia of genes and genomes analysis showed that enrichment pathways were mainly tuberculosis，lysosome，and phagosome pathways.Conclusion In this study，the 11 mRNAs or proteins identified may be core targets for regulating the pathological process of SCI，and the pathological mechanism may be closely related to immune response，lysosome，and phagosome pathways.