Transcriptional Profiling Of Organ Specific Aging In Mice

Vertebrate aging is a complex and progressive process that exhibits organ heterogeneity,with loss of integrity and physiological functions of tissues leading to the impairment of physiology and increasing mortality.In recent years,mice were used as a model to carry out a series of bulk and single-cell transcriptome of different age mice,in order to explore the mechanism of aging.However,there is no description of hippocampus and cortex of neural tissues,and lacks detailed mechanism exploration and cellular and molecular research.This project starts with the transcriptome of the cortex,hippocampus,kidney and heart of young,middle-aged and elderly C57BL/6 mice,to study the universal functional characteristics and tissue-specific changes that occur during aging with various analysis strategies,and with the help of experiments to elucidate the important mechanisms by experiment.The evaluation of quality and volume for the sequencing data showed that,except for the heart samples,the other data met the standard criteria,therefore the data of heart was discarded in the follow-up analysis.The difference of samples was from the distinction of tissue and influence of aging with the results of PCA and MDS algorithm.The CSEPCT was used for exploring the universal characteristics and mechanism of aging.It was found that the neutrophil chemotaxis,base-excision repair-AP site formation and others exist universally for the three tissues during aging.It revealed that chemokine(C-C motif)ligand involved in the neutrophil chemotaxis which associated with senescence-related secretion phenotypes(SASP).And with the GSEA tools,the signature of SASP up-regulated with aging in hippocampus and kidney.In order to explore the specific functional changes of each tissue during aging,the following strategies are adopted: 1)Deseq2 was used to analyze the differentially expressed genes for the three tissues at different age and the Venn diagram was draw;2)four types of trajectory gene clusters were defined for the three tissues during aging: gene clusters that change linearly or approximately linearly with age;that change U-shaped or inverted U-shaped with age;that change significantly in the young and middle-aged stage;that change significantly in the middle-aged stage.3)perform further function research and validation for these clusters.With the analysis of transcriptome,the complement pathway related genes exhibited change with normal aging.C4 b and other complement pathway related genes up-regulated with aging,such as C1 qa,C3 etc.with the results of sequencing and experiment.In order to explore the mechanism of C4 b and the pathway of classical complement,the plasmid of C4 b knock-down was constructed: AAV-Cas Rx-TriplexC4b(treat),AAV-Cas Rx-Triplex-Lac Z(control).The virus was packaged and harvest in the HEK293 T cell,then the high titer virus was transfected into primary neurons and glial cells respectively.It showed that the knock-down system effectively knocked down the m RNA of C4 b,and the dendrites of neuronal morphology was influenced,which analyzed by the cellular immunofluorescence and Sholl analysis.In addition,as the transcription of genes was regulated by transcription factor,the MAGIC algorithm was used to explore the possible transcriptional regulatory mechanism of gene expression changes for three tissues.