Research On The Mechanism Of Interaction Between Breast Cancer Cell And HUVEC Mediated By EVs

Objective:To confirm the effect of MDA-MB-231 and MCF-7 breast cancer cell-derived extracellular vesicles(EVs)on the biological characteristics of human umbilical vein endothelial cell(HUVEC)in vitro experiments,and the effect of HUVEC-EVs on the biological characteristics of MDA-MB-231 and MCF-7 cell.To explore the interaction between the two cells,and to explain the possible mechanism of vascular endothelial cell involved in tumor progression and provide new strategies and ideas for breast cancer treatment.Methods:(1)Breast cancer cell-derived EVs were extracted by differential ultracentrifugation;the morphology of breast cancer cell-derived EVs was observed by transmission electron microscope;the particle size distribution of breast cancer cell-derived EVs was detected by Nanosight;the expression of CD9 and CD63 on the surface of breast cancer cell-derived EVs were detected by Western blot.(2)The internalization of breast cancer cell-derived EVs by HUVEC were observed by immunofluorescence.(3)MTT assay and plate colony formation assay were used to detect the effect of breast cancer cell-derived EVs on the proliferation of HUVEC;the morphological changes of HUVEC after treatment with different concentrations of breast cancer cell-derived EVs were observed;the effect of breast cancer cell-derived EVs on the migration of HUVEC were detected by scratch wound assay and Transwell assay;tube formation assay was to detect the tube formation ability of breast cancer cell-derived EVs on HUVEC;Western blot was used to detect the expression of JAK2?p-JAK2?STAT3?p-STAT3 in HUVEC treated with breast cancer cell-derived EVs.(4)The effect of breast cancer cell-derived EVs on the proliferation of HUVEC pretreated with AG490 inhibitor was observed by plate colony formation;the scratch wound assay and Transwell assay were used to detect the migration of breast cancer cell-derived EVs on HUVEC pretreated with AG490 inhibitor;tube formation assay was to detect the tube formation ability of breast cancer cell-derived EVs on HUVEC pretreated with AG490 inhibitor.(5)Western blot was used to detect the expression of JAK2?p-JAK2?STAT3?p-STAT3 in HUVEC pretreated with AG490 inhibitor.(6)MTT assay and plate colony formation assay were used to detect the effect of HUVEC-EVs on the proliferation of breast cancer cell;the effect of HUVEC-EVs on the migration and invasion of breast cancer cell were detected by scratch wound assay and Transwell assay;Western blot was used to detect the expression of JAK2?p-JAK2?STAT3?p-STAT3 in breast cancer cell treated with HUVEC-EVs.Results:(1)Breast cancer cell-derived EVs were single or clustered membranous nanoscale vesicles with cup-shaped or disc-shaped structure,diameters ranging from tens to hundreds under microscope;Western blot showed that breast cancer cell-derived EVs expressed CD9 and CD63,which were also consistent with the general surface markers of EVs.(2)Immunofluorescence confirmed that breast cancer cell-derived EVs could be internalized into HUVEC.(3)The results of MTT assay,plate colony formation assay,scratch wound assay and Transwell assay showed that breast cancer cell-derived EVs promoted proliferation,migration of HUVEC;when pretreated with AG490 inhibitor,the proliferation,migration of HUVEC decreased;tube formation assay showed that breast cancer cell-derived EVs promoted the tube formatin ability of HUVEC;when pretreated with AG490 inhibitor,the tube formatin ability of HUVEC decreased.(4)Western blot showed that when treated with increase of the concentration of breast cancer cell-derived EVs,p-JAK2 and p-STAT3 expression in HUVEC increased;when pretreated with AG490 inhibitor,p-JAK2 and p-STAT3 expression in HUVEC decreased.(5)The results of MTT assay,plate colony formation assay,scratch wound assay and Transwell assay showed that HUVEC-EVs promoted proliferation,migration and invasion of breast cancer cell.(6)Western blot showed that when treated with increase of the concentration of HUVEC-EVs,p-JAK2 and p-STAT3 expression in breast cancer cell increased.Conclusions:Breast cancer cell-derived EVs promote the proliferation,migration,and tube formation ability of HUVEC through the JAK2/STAT3 pathway.Similarily,HUVEC-EVs may also promote proliferation,migration and invasion of breast cancer cell through the JAK2/STAT3 pathway.The results of this study further confirm that blocking tumor angiogenesis is one of the effective ways to treat breast cancer.