SENP1 Reduces Stemness Of Osteosarcoma And Increases HSVtk/GCV Sensitivity

Objective:Explore the role of SUMO1 and SENP1 in the development of osteosarcoma;Clarify the role of SUMO1 and SENP1 in the maintenance of osteosarcoma cells,and the effect on the sensitivity of traditional chemotherapy,in order to provides a new therapeutic strategy for the treatment of osteosarcoma stem cells that are not sensitive to chemotherapy.Methods:(1)(1)18 osteosarcoma patients were collected and the total protein was extracted,The expression of SUMO1 and SENP1 in osteosarcoma was detected by immunoblotting.(2)Western Blotting was used to detect the expression of SUMO1and SENP1 in hFOB1.19 and 143B,MG-63,and U-2OS.(3)CD133 antibody was labeled with 143B,and CD133~+cells were sorted by flow cytometry.CD133~+cells and CD133~-cells were cultured separately to detect differences of SUMO1 and SENP1 expression.(4)CD133~+osteosarcoma stem cells were in the Quiescent,proliferative and senescence phase of the cell cycle,and the expression of SUMO1and SENP1 and stem maintenance related genes Oct4,HIF-1?and PCNA were detected by immunoblotting.(2)(1)RNA interference silenced SUMO1 in CD133~+cells,observed the ability of cloning,Ki67 staining to detect cell proliferation,flow cytometry to detect CD133~+population ratio and cell cycle,transwell to detect cell migration,and four proteins PCNA,Oct4,HIF-1?,Aktl,which have been identified as SUMO1 target proteins,two migration-related proteins MMP2,MMP9 and apoptosis-related protein Caspase-3 were detected.(2)SENP1 was analyzed in overexpressing SENP1 CD133~+to observe the stem cell characteristics and function of osteosarcoma stem cells.(3)Transfecting tk,SENP1,tk+SENP1 gene,ganciclovir GCV treatment,analysis cell apoptosis and the sensitivity to tk/GCV in vitro.(4)Constructing a subcutaneous xenograft tumor model:control group;SENP1 group;HSVtk/GCV group;combined group.Observed tumors growth volume,mass,situ apoptosis,SUMO1,SENP1 and PCNA proteins expression.Results:(1)(1)SUMO1 of osteosarcoma was significantly higher than adjacent tumor tissue;SENP1 decreased by about 20%.(2)SUMO1 in 143B,MG-63,and U2O-S was significantly increased compared with hFOB1.19,and SENP1 was significantly decreased.(3)The expression of SUMO1 in CD133~+tumor stem cells was higher and SENP1 protein expression was lower.(4)Compared with proliferative or senescent cells,SUMO1 in osteosarcoma stem cells at quiescent stage was significantly increased,SENP1 was lower,Oct4 and HIF-1?were significantly higher while PCNA were not increased.(2)(1)siRNA-SUMO1 in CD133~+cells,the morphology of tumor stem cell failed to clone spheres,adherent phenomenon occured,the volume of cloned spheres was significantly reduced,and migration cell was significantly reduced.Flow cytometry detected CD133~+population proportion decreased significantly,and most of the cell cycle changed from G0/G1 phase to S/G2 phase,Ki67 decreased.PCNA,Oct4,HIF-1?,MMP2,MMP9,and Caspase-3 decreased while Akt1 was not decreased.(2)SENP1 was overexpressed in CD133~+cells,it can be seen that the surrounding debris is fragmented,the ability to form a cloned sphere is reduced.The cell cycle is mostly changed from G0/G1 phase to S/G2 phase,and the expression of Ki67 is decreased.The cell migration rate only changed slightly,and the Ki67positive rate only increased slightly.PCNA,Oct4,MMP9,HIF-1?and Caspase-3protein decreased but MMP2 and Akt1 not obviously.(3)HSVtk/GCV significantly increased the ratio of LDH and apoptotic cells,SENP1 alone had only a slight effect.When combined HSVtk/GCV with the SENP1,apoptosis increased significantly.(4)In animals,SENP1 alone showed no significant tumor diameter,volume,mass,apoptosis change.HSVtk/GCV alone can reduce the above indicators to a certain extent,inhibit tumor growth,but can not completely eliminate the tumor.The combination of SENP1 and HSVtk/GCV further reduced tumor volume,mass and induced apoptosis.Conclusions:(1)SUMO1 in osteosarcoma was significantly increased,SENP1 was decreased,and more significant in osteosarcoma stem cells;(2)Silencing SUMO1 induces rapid aging of osteosarcoma stem cells,inhibits cell proliferation and migration,and induces a large number of apoptosis;(3)SENP1 significantly reduced the stem cell dryness maintenance ability,only slightly affecting osteosarcoma stem cell migration and apoptosis;(4)SENP1 significantly increased the sensitivity of osteosarcoma to HSVtk/GCV.