K15 Protein Of Kaposi's Sarcoma Herpesviruses Increases Endothelial Cell Proliferation And Migration Through Store-operated Calcium Entry

Objective : to investigate the effects of?KSHV?K15 protein of kaposarcoma associated herpesvirus on the proliferation and migration of vascular endothelial cells and smooth muscle cells,and the relationship between these effects and the manipulation of calcium influx?SOCE?by calcium storage.At the same time,the molecular mechanism of K15 protein promoting cell proliferation and migration was studied.Methods:?1?preparation of lentivirus particles: using recombinant plasmids pFJ-K15 P and K15 P mutants pFJ-K15P?YF?as template,specific primers were designed to amplify KSHV K15 P and K15P?YF?gene fragments by PCR.Construction of T vector and lentivirus expression vector pHAGE-CMV-MCS-K15P-Puro,pHAGE-CMV-MCS K15P?YF?Puro,by restriction endonuclease Not I and BamH I digestion and TA cloning The constructed lentivirus expression vector was co-transfected with two auxiliary packaging plasmids PSPAX2 and pMD2.g into HEK 293 T cells by lip2000.The lentivirus was collected for preservation after 48 hours.?2?Endothelial cell infection and screening of stable expression strain: lentivirus infected human umbilical vein endothelial cell?EA.hy926?and stable expression strain,Western blot was used to detect the expression of K15 P and K15P?YF?protein.?3?the relationship between K15 protein and SOCE: human umbilical vein endothelial cells?EA.hy 926?were infected with lentivirus particles,which activated SOCE,by calcium agonist.The changes of intracellular calcium concentration in lentivirus control group,lentivirus K15P?YF?group and lentivirus K15 P infected endothelial cells were detected by Leica TCS SP5 laser confocal system.Western blot was used to detect the expression of STIM1 and Orail1,two of the most important protein families of SOCE.?4?K15 protein and cell proliferation and migration and its molecular mechanism: CCK-8 kit was used to detect the proliferation of EA.hy 926 cells induced by three lentiviruses.Cell migration assay?Transwell?to observe the changes of cell migration induced by three lentiviruses infected EA.hy 926 cells and?HUVSMC?migration of human umbilical vein smooth muscle cells co-cultured with three lentivirus infected EA.hy 926 cells.Western blot was used to detect endothelium,The effects of three lentivirus infected EA.hy 926 cells on the expression of HUVSMC ?-smooth muscle actin??-SM-actin?in smooth muscle cell coculture system were studied.The proliferation of HUVSMC was detected by CCK-8 kit.Results:?1?Recombinant plasmids containing K15 P and K15P?YF?fragments were successfully constructed by restriction endonuclease digestion and sequencing.The recombinant lentivirus vector and two auxiliary plasmids PSPAX2 and pMD2.g were co-transfected with liposome transfection reagent to produce lentivirus particles in HEK 293 T cells.?2?Lentivirus infected EA.hy926 cells.The stable expression of K15 P protein in EA.hy926 cells infected with purine mycin could be detected by survival,Western blot in the culture medium containing purine mycin.?3?In EA.hy 926 cells,compared with empty vector,the intracellular Ca ^{2 +}concentration,Ca ^{2 +}release and SOCE were significantly increased in K15P?YF?cells.Western blot showed that the expression of Orail1 protein in K15 P cells was significantly higher than that in the other two groups.?4?In EA.hy 926 cells,the proliferation and migration of K15 P group were significantly higher than those of control group and K15 P mutant group.The expression of ?-SM-actin in HUVSMC,cells co-cultured with EA.hy 926 cells expressing K15 P was decreased,and the ability of proliferation and migration was also enhanced.Conclusion: The lentivirus expression vector was successfully constructed and the lentivirus system expressing K15 P and its mutants was established.Among the endothelial cells stably expressing K15 P protein,K15 P could promote the proliferation and migration of endothelial cells and promote the proliferation and migration of endothelial cells to co-cultured smooth muscle cells.K15 P could increase the intracellular calcium concentration of endothelial cells.Reduction of endoplasmic reticulum calcium release and enhancement of SOCE.