The Role And Mechanism Of Platelet Exosomal LRG1/OLFM4 In The Progression Of Multiple Myeloma

Background: Multiple myeloma(MM)is a malignant disease derived from plasma cells,which has a high morbidity and mortality.In recent years,with the emergence of new treatment protocols and the improvement of various chemotherapy protocols,the median survival time and prognosis of MM patients have greatly improved.However,there are still a large number of patients with serious deterioration of the condition,or recurrence and drug resistance after treatment.Further research on the mechanisms of its occurrence and development to improving patient survival rate and prognosis is still an urgent problem to be solved.Platelets are derived from mature megakaryocyte in bone marrow hematopoietic tissue,and play an important role in many other pathophysiological processes in addition to the main hemostatic function.Platelet activation is closely related to the progression of tumors.Platelet activation in tumor is accompanied by the release of a large number of exosomes,which has great potential in early diagnosis and treatment of diseases.Exploring the role and mechanism of exosomes released by activated platelets in the progression of MM will provide new ideas for the diagnosis and treatment of MM.Objective: This study aims to explore the key functional molecules in platelets that may participate in the progression of MM,and explore their specific modes of action and mechanisms through in vitro and in vivo experiments.Method:(1)Peripheral blood of healthy volunteers and MM patients was collected,and the platelet activation state was observed by flow cytometry,platelet aggregation analysis and electron microscopy.The influence of platelet on MM cell proliferation and apoptosis was detected by EdU,flow cytometry and WB.(2)Quantitative proteomics combined with bioinformatics analysis screened out the key functional molecule leucine-rich-alpha-2-glycoprotein 1(LRG1)in activated platelets that may be involved in the progression of MM.Its biological function was explored through label tracing,WB,EdU,flow cytometry and other methods,and its correlation with the prognosis of MM was analyzed in clinical samples.(3)Protein interaction and Co-Immunoprecipitation were used to explore the downstream molecules of LRG1,and the specific mechanism of LRG1 on MM was explored through WB,immunofluorescence,immunohistochemistry,angiogenesis experiments and mouse subcutaneous tumor bearing model.Result:(1)Compared with healthy volunteers,the platelet activation rate was higher in MM patients.Under electron microscopy,morphological changes and the extension of pseudopodia in platelets were observed,and ADP induced platelet aggregation rate was higher.Platelet rich plasma(PRP)from MM patients can promote MM cell proliferation and inhibit MM cell apoptosis.(2)Quantitative proteomics and bioinformatics analysis revealed 477 differentially expressed proteins in PRP between healthy volunteers and MM patients,which were closely related to vesicle formation,EMT and angiogenesis.WB confirmed that LRG1 was highly expressed in PRP of MM patients,and LRG1 was significantly enriched in platelet exosomes of MM patients.Analysis of clinical samples showed that high expression of platelet exosomal LRG1 was a factor for poor prognosis in MM patients.Platelet exosomal LRG1 in MM patients promote proliferation and inhibit apoptosis of MM cells.(3)Protein interaction analysis showed that LRG1 interacted with olfactomedin 4(OLFM4),and co-immunoprecipitation confirmed that they were bound to each other through LRR and olfactomedin domains.Overexpression of LRG1 promotes EMT and angiogenesis in MM,and siRNA interference with OLFM4 reverses this effect.Animal experiments showed that overexpression of LRG1 increased the size of subcutaneous tumors in tumor-bearing mice,promoted cell proliferation and inhibited apoptosis in tumor tissues.In addition,more vascular like structures are formed in tumor tissue,and more VEGF is secreted.siRNA interference with OLFM4 reversed this growth-promoting effect.Conclusion: This study found the role of activated platelet derived exosomal LRG1 in promoting the disease progression of MM and its correlation with the poor prognosis of MM for the first time,and revealed that platelet exosomal LRG1 promotes EMT and angiogenesis of MM through interaction with OLFM4,and affects the progress of MM.26 figures,8 tables,140 references.