Background
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).
Methods
High-throughput sequencing was carried out to detect the cargos of exosomes from clinical serum and U266 cells, then GSE5900 database was analysed 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 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. Furthermore, network pharmacology was employed to 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.
Results
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, and the tube formation could be blocked if we knockout 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 confirmed by luciferase reporter assay. The docking site was determined between PF and MEF2A of MALAT1, then the effect of PF on MEF2A/MALAT1 was confirmed by WB or PCR test.
Conclusion
To summarize, our study revealed that myeloma cells can increase the angiogenesis by release 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 intervene the MEF2A/MALAT1 in myeloma cells.
