The Effect And Mechanism Of SPHK1/S1P- SIRT1 On The Characteristic Of HUVECs

Objective:The study aimed to investigate the effect of SPHK1/S1P and SIRT1 on the characteristic of HUVECs and clarify the relationship and mechanism between SPHK1 and SIRT1.Methods:Using digestion, recovery, connection and other molecular biological methods to build the lentivirus vector which carried SPHK1 interfering sequence and green fluorescence proteins (GFP). Proliferation was detected via CCK8; Migration was detected by Transwell; Apoptosis and cell cycle was detected by flow cytometry; Forming of tubular structure was detected by matrigel; Gene expression of SPHK1 and SIRT1 was detected by RT-PCR, protein expression of SPHK1 and SIRT1 and the phosphorylation of ERK, AKT, P38 MAPK and NF-KappaB was detected by Western Blot.Results:We successfully constructed the lentivirus vector PLKO.1-shSPHKl-GFP which carried SPHK1 interfering sequence and GFP. The results showed that interfering SPHK1 could significantly reduce the gene and protein expression of SIRT1, but interfering SIRT1 couldn’t affect the gene expression of SPHK1. SIP which is the catalytic products of SPHK1 could increase both the gene and protein expression of SIRT1. Furthermore, the research indicated that interfering SIRT1 could significantly reduce the proliferation and migration of HUVECs, and it also could promote the apoptosis and the ability to forming the tubular structure of HUVECs. But interfering S1RT1 couldn’t affect the cell cycle of HUVECs. Similarly, interfering SPHK1 could decrease the proliferation and migration of HUVECs. SIP could significantly increase the proliferation and migration of HUVECs, but it couldn’t affect the apoptosis, cell cycle and the formation of tubular structures of HUVECs. Interestingly, SIP could reverse the declined proliferation and migration causing by interfering SIRT1. Western Blot showed that the phosphorylation of ERK, P38-MAPK and AKT were all decreased after interfering SPHK1. Using PD98059 inhibit ERK pathway, SB203580 inhibit P38-MPAK pathway, Wortmannin inhibit AKT pathway, the protein expression of SIRT1 and the migration of HUVECs were all significantly reduced.Conclusions:The effect of SIRT1 on the characteristics of HUVECs could be mediated by SPHK1 via ERK, P38-MAPK and AKT pathway. We found a new signal pathway of SPHK1/S1P-ERK/P38-MAPK/AKT-SIRT1 at the first time.