Exploration Of GART Promoting The Cellular Proliferation In Multiple Myeloma And Related Mechanism

Background and ObjectiveMultiple Myeloma(MM)is a neoplastic plasma cell disease and is a hematological malignancy.MM develops from monoclonal gammopathy of undetermined significance(MGUS)with clinical symptoms such as hypercalcemia,anemia,and renal failure.Although a variety of chemotherapy drugs have been used in clinic,for instance bortezomib,thalidomide,etc,which have good curative effect on MM,it still cannot be completely cured as reason of the recurrence and drug resistance are one of the main reasons.Therefore,to explore the mechanism of MM recurrence and to find a method that can effectively intervene in MM is one of the problems that needs to be solved urgently.Our research group has found that GART(phosphoribosylglycinamide formyltransferase)is involved in the regulation of MM disease process through protein chip and clinical data analysis of MM patients.GART is a folate metabolizing enzyme involved in the purine synthesis pathway.It has been reported that the GART-mediated de novo purine synthesis pathway is abnormally active in malignant tumors,and the high expression of GART is closely related to the poor prognosis of MM patients,but its specific regulatory mechanism in malignant tumors,especially in MM,has not been identified.This project intends to study the role of GART in the proliferation of MM cells in vitro and in vivo,to explore the specific mechanism of GART in affecting the proliferation of MM cells,and to provide new molecular targets for the clinical diagnosis and treatment of MM.Methods1.Using GEPIA,TT2,APEX and other databases,analyzed the survival curves of normal people and MM patients,and explored the relationship between GART expression and poor prognosis of MM patients.2.Lentiviral transfection was used to construct stable transfected GART overexpression and knockdown MM cell lines and the transfection efficiency was verified by Western blot(WB),the proliferation of MM cells was detected by CCK-8 and soft agar cloning assay,the mitotic cycle and apoptosis of MM cells were detected by flow cytometry.3.The effect of GART inhibitor on the proliferation of MM cells,pemetrexed(PEM),was detected by CCK-8 assay,the effect of PEM on the apoptosis of MM cells was used by flow cytometry,an immunodeficient mouse xenograft tumor model was constructed to observe the effect of GART overexpression on tumor growth in vivo,and to explore the effect of PEM on tumor proliferation.4.Co-immunoprecipitation(Co-IP)was used to enrich GART-binding proteins,HSP90α were found by protein profiling analysis,and their binding was verified by Co-IP,immunofluorescence assay was applied to determine the co-localization of GART and its downstream proteins in MM cells,the modification of HSP90α by GART was demonstrated by LC-MS,and ATPase activity assay was used to detect the effect of GART on the ATPase activity of HSP90α.Results1.The GEPIA database analysis result showed that GART was highly expressed in a variety of tumors,the clinical MM-related database analysis revealed that the high expression of GART is closely related to the decreased survival rate of MM patients.2.The finding of WB proved that the stable transfected MM cell lines were successfully constructed.The results of CCK-8 and soft agar cloning assay both showed that overexpression of GART promoted the proliferation of MM cells,and knockdown of GART inhibited cell proliferation,flow cytometry results showed that overexpression of GART increased the ratio of G2/M phase in mitosis of MM cells,and knockdown of GART accelerated apoptosis of MM cells.3.It was found that PEM could inhibit the proliferation of MM cells via CCK-8 assay,and promote MM cell apoptosis through flow cytometry.The xenograft tumor model in immunodeficient NOD/SCID mice demonstrated that overexpression of GART significantly promoted tumor growth,while the application of PEM effectively reversed tumor growth.4.The analysis of protein profiles suggested that HSP90α might be involved in GART promoting the proliferation of MM cells,Co-IP experiments confirmed the interaction between GART and HSP90α,immunofluorescence staining exhibited that GART and HSP90α colocalized in MM cells,LC-MS showed that the E229 site of HSP90α could be methylated by GART,Methylated HSP90α affected its ATPase activity.Overexpressed GART increased the ATPase activity of HSP90a,while down-regulated GART blunted its ATPase activity.After treatment of PEM,the ATPase activity of HSP90a was significantly reduced.ConclusionThis present study has determined the effect of GART in promoting MM proliferation.Mechanically,GART may modify HSP90a through methylation,thereby increasing the ATPase activity of HSP90α,and ultimately promoting the proliferation of MM cells.This study intends to provide new molecular targets for the diagnosis and treatment of MM.