| Cardiovascular disease is now the leading cause of death worldwide. Recent clinical studies have found that a new type of post-translational modification SUMOylation has a close relationship with the occurrence and development of many kinds of cardiovascular diseases. This study explored the effect and mechanism SUMOylation works on As (atherosclerosis) from a kind of warning molecules "Alarmin" which are released by activated vascular endothelial cells, and found the mutations of deSUMOylated enzyme in SUMO gene family which are associated with As by genetic polymorphism marked method, to provide new theoretical basis and new strategies for interventional therapy of human cardiovascular disease.First we collected a large amount of clinical blood samples of As patients and healthy people, and confirmed the level difference of Alarmins (IL-1, IL-18, IL-33, HMGB1) in plasma by ELISA, the result showed that the patients with As less than 50% had the highest mean concentration of Alarmins in plasma, which was significantly different from the other two groups (P<0.05). According to the clinical evidence, we cultured vascular endothelial cells in vitro and exerted them H2O2, TNF-a and IFN-y treatment within 24 hours to simulate the clinical pathological state. By a series of qRT-PCR, Western Blot, ELISA tests, we found that the mRNA level of proteins in SUMO family and Alarmins had no significant change (P>0.05) in stimulated cells, while the level of SUMOylation and the concentration of extracellular Alarmins raised at the same time and reached the maximum at a certain time (P<0.05 except a few individuals, the difference was significant), which verified the functional mechanism of SUMO connecting the target proteins directly for modification and pro-Alarmins release after machining as well. Then we discovered that, in cells that SUMO1/SUMO2 (SUMOylated molecule)/UBC9 (SUMOylated E2 enzyme) gene was knocked down, the expression of Alarmins was significantly suppressed and the content of Alarmins (including their initial expression level) inside and outside cells was lower than those in normal cells (P<0.05 except a few individuals, the difference was significant); while the conclusion was just the opposite in cells that SENP1 (deSUMOylated enzyme) gene was knocked down (P>0.05), which proved the positive correlation between SUMOylation and the expression of Alarmins. Furthermore, we examined the main proteins NLRP3, ASC and Caspase-1 of inflammasome which mediates the release of Alarmins, and found that the expression of ASC and NLRP3 was not changed with the stimulus of pathological condition, they bound directly and caused high concentration of pro-Caspase-1, and then cut it to Caspase-1, so as to promote the release of Alarmins.In addition, we used the clinical blood samples of As patients and healthy people as the experimental material and screened out three heterozygous mutation sites c.220G>T, c.504T>C (rs886588), c.1211A>G of SENP1 by the first generation DNA sequencing technology and contrasting with databases. Among them, c.220G>T and c.1211A>G were not reported SNPs (Single Nucleotide Polymorphisms) before and c.220G>T mutation was statistically significant (f=0.30). Our study tentatively proved that the three mutations could shorten the half life of SENP1 and varied its localization to the cytoplasm, and the superimposed effect of the three mutations was stronger, but none of those could inactivate SENP1 protease.In summary, SUMOylation had positive function on the expression and release of Alarmins, and the mutation sites c.220G>T, c.504T>C, c.1211A>G of SENP1 changed its stability and localization. However, the specific mechanism of how SUMO regulates Alarmins, the functional effect of the mutation of SENP1 in vitro and in vivo, and their contribution to the pathogenesis of As remains to be further studied. |
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