Paeoniflorin inhibits angiogenesis in multiple myeloma by decreasing the MEF2A level to downregulate the expression of lncRNA MALAT1 within exosomes

Angiogenesis is a pivotal mechanism driving tumor proliferation, and the epigenetic regulation of angiogenesis represents a cutting-edge area of current research in multiple myeloma (MM). High-throughput sequencing was carried out to detect the cargos of exosomes from clinical serum and U266 cells, then GSE108824 database was analyzed for the finding of differentially expressed genes (DEGs). The intersect set was made based on the three gene sets. The clinical features of Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1)were verified through GEO and clinicopathological data analyses. Cell viability, tube formation assay, level of MALAT1 and VEGFA were used to evaluate the effect of U266 exosome pretreated with or without paeoniflorin (PF) on angiogenesis in HUVEC cells. Subcutaneous tumor-bearing mice were established by injection of U266 cells and exosomes derived from U266 cells which pretreated with or without PF. Tumor size, HE staining, analysis of MALAT1 and VEGFA levels, as well as IHC staining for VEGFA, CD31, and Ki67 were performed to evaluate the in vivo effects of PF. The interactions between MALAT1, VEGFA, and microRNAs were demonstrated by TargetScan, MiRanda databases and Luciferase reporter assay. Furthermore, network pharmacology and RROMO, Genecards, AnimalTFDB, JASPAR databases were combinedto predict transcription factors (TF) associated with MALAT1 and analyze the binding sites between PF and these transcription factors. The validation of PF effect on TF was conducted by WB and PCR. Clinical studies indicated a notable positive correlation between MALAT1 level and VEGFA, CD31 expression, moreover, the high MALAT1 level is closely related to poor prognosis of MM. We demonstrated that MALAT1 was the highest expression linear RNA in U266 exosomes and could be transported to HUVEC cells through exosomes, promoting HUVEC cells differentiation and angiogenesis by stimulating VEGFA expression. The tube formation could be blocked if we knockdown the MALAT1 in U266 exosome. It was also proved that this pathological process can be blocked by PF in vitro and in vivo experiments. The ceRNA mechanism in MALAT1/miR-17/VEGFA was predicted and then confirmed by luciferase reporter assay. 2548 PF target genes were retrieved from databases, and the intersections with MALAT1-related differentially expressed proteins, mRNA and TF gene were identified Venn diagaram. MEF2A binding sites were predicted JASPAR, finally molecular docking showed strong affinity between PF and MEF2A (-16.5 kcal/mol).Then the effect of PF on MEF2A/MALAT1 was confirmed by WB or PCR test. To summarize, our study revealed that myeloma cells can increase angiogenesis by releasing exosome to influence the endothelial cells. The MALAT1 from myeloma cells is the crucial factor in this pathological process. PF can obstruct this process by intervening in the MEF2A/MALAT1 in myeloma cells.