• Determining expression levels of microRNAs involved in angiogenesis (including miR-126, miR-132 and miR-296) in exosomes derived from MDA-MB-231 breast tumor cells and investigating the effects of exosome treatment on VEGF protein secretion from endothelial cells.
  • Maryam Sayyad,1,* Sadegh Babashah,2 Seyad Hadi Mousavi,3
    1. Islamic azad university pharmaceutical scienses branch
    2. Tarbiat Modares University
    3. Tehran University Medical Sciences


  • Introduction: Breast cancer is recognized as a major clinical challenge in the context of women's illnesses around the world. Since cancer cells are anxious for the survival and development of the metabolites and growth factors required to activate the pathogenesis of angiogenesis, studies are underway in the context of a process that causes the tumor to induce angiogenesis of endothelial cells creating new blood vessels is important. Therefore, finding the activator pathways and cellular components that enable the tumor to progress from the angiogenesis is of critical importance. Exosomes are small vesicles derived from multi-vesicular bodies, which are secreted by many cells into an out-of-cell environment and thus contribute to target cells through intercellular communication through the transmission of genetic information such as coding and non-coding RNAs. Tumor-derived exosomes are considered as a rich source of microRNAs that can regulate the function of other cells in the tumor micro-environment, including endothelial cells of the vascular. However, the exact mechanisms by which tumor-derived exosomes affect the micro-environment cells and the biological function of exosome microRNAs in receptor cells are not well defined.
  • Methods: MDA-MB-231 breast cancer cells, Human umbilical vein endothelial cells (HUVECs) Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 1% antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) (all from Gibco BRL, Rockville, MD, US) at 37°C in a humidified atmosphere of 5% CO2. • Cell culture supernatants were harvested and centrifuged at 300 g for 15 min. • ExoQuick-TC Exosome Precipitation Solution was added to the supernatants and the mixture was refrigerated overnight. • The mixture was centrifuged at 15000 g for 30 min • The exosome pellet was resuspended in PBS .For characterization of purified exosomes. We used Scanning electron microscopy (SEM) and Dynamic light scattering measurement • Total RNA extraction by Trizol and Expression Analysis of miRNA by Q-RT-PCR • Quantitative measurement of secreted VEGF were performed on the supernatants of HUVECs treated with tumor-derived exosomes. And ELISA showed levels of secreted VEGF from exosome-treated endotelial cells.
  • Results: SEM revealed that the purified exosomes had a spherical shape with a diameter of about 40-100 nm. And Measurement of exosome size by DLS number analysis showed a single bell-shaped size distribution with peak of ~60 nm. The concentration of total RNA was measured by a spectro photometer and the result of samples indicate a good quality of RNA. Determination the Expression level of miR-126 miR-132 and miR-296as a proangiogenic miRNA in exosomes and their corresponding donor cells by Q-RT-PCR showed increased levels of proangiogenic miRNAs miR-126, miR-132 and miR-296 in exosomes derived from breast tumor cells, It was also found that tumor cell-derived exosomes have a significant effect on increased vascular endothelial growth factor expression (VEGF) expression in a time-dependent manner in endothelial cells. (*** P<0.001)
  • Conclusion: This study suggests that the exosomal transfer of proangiogenic miRNAs to endothelial cells may be a mechanism for describing how the paracrine function of the tumor-derived exosomes Through which these microvesicles regulate vascular behavior in the micro-environment of cancer cells.
  • Keywords: Breast cancer, Exosome, Tumor angiogenesis, VEGF