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

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.