Study of the effect of hypoxia on proliferative and migratory functions of exosomes derived from metastatic breast cancer cell line on endothelial cells (HUVEC)

Abstract

Hypoxia is a non-physiological level of oxygen pressure and a prevalent phenomenon in most of the tumors. It also results in an unusual expansion of tumors through a variety of ways, such as angiogenesis. There are different mechanisms of angiogenesis for cancerous cells, including breast cancer, to expand the tumors which are one of the mechanisms of secretion of exosomes, containing oncogenes and miRNA that are influential in the expression of these factors. The objective of this study is to determine the effect of hypoxia or the change in the content of exosomes secreted from the cancerous breast cells of MDA-MB-231 and BT-474. It also aims to determine the effect that these exosomes have on the function of endothelial cells HUVEC concerning proliferation, immigration and gene expression connected with immigration and proliferations like HIF-1α and TGF-β and expression of 16 and 433 miRNA. Materials and Method: cellular type MDA-MB-231 and BT-474 were cultured in a complete cell culture environment under the hypoxia and non-hypoxia conditions. After 24 hours, the surface liquid was collected to extract exosomes. The exosomes in the surface liquid were extracted using ultra centrifuge. The exosome extraction was approved by means of a electron microscope photography. HUVEC cells were cultured as a cellular model of endothelium and were sided in 96-well plates and were exposed to the effect of different concentration of extracted exosomes. Through this, the effect of concentrations of exosomes on the proliferation of HUVEC cells was measured. Then, in order to do scratch test, HUVEC cells were sided in 24-well plates and the effect of exosomes of MDA-BT-474 and MB-231 cells on the immigration of these cells was measured. After that, the RNA of HUVEC calls were extracted under the effect of exosomes and CDNA were synthesized and the expression of mRNA and controlling miRNA, the expression of gens connected with immigration and proliferation of gens such as HIF-1α, VEGFR, and miRNA 16 and miR-433 were measured through qAR-PCR test. Results: the findings of the study on the cell types MDA-MB-231 and BT-474 indicated that the effect of secreted exosomes from this type of cells resulted in the hypoxia to increase the proliferation and immigration HUVEC cells.