Mechanistic Study Of NSD2 In Endothelial Cell Senescence

Due to the intrinsic complexity of the aging process,there has been great interest in uncovering the underlying causes and factors associated with aging.Over the past decades,scientific studies have attempted to determine the effects of genetic regulation and environmental changes on aging.A growing body of evidence suggests that epigenetic alterations can be considered as markers of aging.The aging process involves many epigenetic factors,such as histone modifications,DNA methylation and changes in chromatin structure.Histone modifiers play a crucial role in gene expression and metabolic regulation.however,the knowledge of how histone modifiers regulate cellular senescence is still limited.This thesis is focused on the effects of histone modifying factors on cellular senescence.Using human umbilical vein endothelial cells(HUVECs)as a model,transcriptome sequencing analysis was performed in cells with different degrees of(replicative)senescence,and 32 significantly altered histone methylation modifying enzyme genes were screened from the 2330 genes that were down-regulated due to senescence,of which the expression of 16 genes gradually decreased during cellular senescence.And the expression of 16 genes was inhibited by RNAi interference,and it was found that inhibition of histone the expression of methyltransferase NSD2 was found to significantly promote cellular senescence,and the role and mechanism of NSD2 affecting endothelial cell senescence were further elucidated.The contents of this thesis and findings are as follows:1.We successfully isolated HUVECs and passaged in vitro cultures to generate replicative senescence models.P6,P20,and P34 cells with presumed different degrees of senescence were selected and transcriptome sequencing was performed.Data analysis revealed that replicative senescence resulted in upregulation of 1707 genes and downregulation of 2330 genes.2.32 significantly altered histone methylation modifying enzyme genes were screened out from the 2330 down-regulated genes,16 of which were progressively decreased with replicative cellular senescence.Inhibition of expression of those 16 genes through RNAi interference as well as further screening of genes associated with cellular senescence using SA-β-Gal staining showed that knockdown of NSD2 expression in endothelial cells increased the rate of positive SA-β-Gal staining by 63.4%(p<0.001).3.To further investigate the role of NSD2 deletion on cellular senescence.We found that the mRNA and protein expression of NSD2 was significantly decreased during endothelial cell senescence(p<0.001),as the NSD2 expression was significantly lower in senescent cells than in young cells.Knockdown of NSD2 expression in endothelial cells significantly inhibited the proliferative capacity of cells(p<0.001),affected cell cycle progression,blocked some cells in G0/G1 phase,and led to a 57.7%increase in the proportion of Senescence-associated heterochromatin foci(SAHF)positive cells(p<0.001).In the in vitro tubule formation assays,knockdown of NSD2 resulted in a 32.2% reduction in tubule length(p<0.01),which significantly affected vascular regeneration function.The above results suggest that knockdown of NSD2 expression in endothelial cells can significantly promote endothelial cell senescence.4.To explore the upstream signaling pathway of NSD2 regulating cellular senescence.Eight miRNAs targeting binding to the 3’ UTR region of NSD2 mRNA were obtained by miRNA prediction database,and the expression of the eight miRNAs was detected in different replicative senescence P6,P20,and P34 cell models,where only miR-34a-5p was found to gradually increase with the replicative senescence of cells.The dual-luciferase assay showed that miR-34a-5p could directly target the 3’UTR region of NSD2 mRNA to interfere with gene expression.And subsequent experiments also revealed that miR-34a-5p downregulated NSD2 protein expression by47.8%(p=0.049).Overexpression of miR-34a-5p promoted endothelial cell senescence,and inhibition of miR-34a-5p delayed endothelial cell senescence.Those results suggest that miR-34a-5p can directly target NSD2,inhibit NSD2 expression,and promote endothelial cell senescence.5.To further investigate the downstream mechanism of NSD2 regulation of cellular senescence.By examining the effect of NSD2 knock down or miR-34a-5p overexpression in endothelial cells on H3K36me2 levels,it was found that the overall level of H3K36me2 was significantly downregulated in the NSD2 knocked down as well as miR-34a-5p overexpressing HUVECs(p<0.01).6.Transcriptome sequencing of NSD2 knocked down HUVECs revealed that knockdown of NSD2 results in upregulation of 604 genes and downregulation of 827 genes.We cross-tabulated the genes regulated by replicative senescence with those regulated by knockdown of NSD2 and found 383 co-regulated genes,which were mainly enriched in the immune response pathway.In summary,this study is the first to report that knockdown of histone methyltransferase NSD2 promotes endothelial cell senescence,identifies miR-34a-5p as its upstream regulator as well as a new signaling pathway axis that promotes endothelial cell senescence by reducing histone H3K36me2 through down-regulation of gene NSD2.This study provides the basis for the effect of histone modifying factors on endothelial cell senescence and provides a new potential target for delaying cellular senescence and thereby achieving healthy longevity.