• MiR-34a-loaded extra cellular vesicles derived from 4T1 cells, effectively reduced 4T1 cell migration capacity time-dependently
  • Mahsa Hajivalili,1,* Maryam Hosseini,2 Bahare Niknam,3
    1. Immunology Research Center, Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Tehran, Iran
    2. Trauma Research Center, Emtiaz Trauma Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
    3. Departement of Immunology, School of medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran


  • Introduction: Changes in tumor suppressor miRNAs is one of the leading cause of cancer progression. Mir-34a is a well-known anti-tumor miRNA. An obstacle for therapeutic application of this miRNA is proper delivery vehicle. Characteristics of small extracellular vesicles (sEVs) make them a favored vehicle for delivering of therapeutic agents. As in triple negative breast cancer, finding an ideal therapeutic method is a real challenge, we aimed to apply tumor derived sEVs (tsEVs) for miR-34a delivery in 4T1 cells and evaluated its anti-migratory effects.
  • Methods: 4T1 cells were cultured under proper cell culture condition and after reaching to a an approximate of 80% confluency, FBS-free medium was added to the cells and after 48 hours, the conditioned media was gathered for tsEV purification by commercial kit. The purified tsEVs were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS) and bicinchoninic assay (BCA). To load miR-34a, modified CaCl2 method was conducted. Confirmation of loading was done by Real-time PCR method.4T1 cells were cultured in 6 well plate and after 24 hr a scratch was created by a 200μl pipette tip, afterwards each well was treated by 25μg/ml of tsEVs and miR-34a-tsEVs, respectively. Untreated cells were considered as control group. An image was taken from each treated group after 0, 6, 12 and 24 hr. The images were analyzed by image J software according to percentage of re-filled scratch area. Statistical analysis was performed by ANOVA test and Graph pad Prism 8 software.
  • Results: Our data showed that treatment with miR-34a-tsEV significantly reduced migration capacity of 4T1 cells in a time-dependent manner, which the highest scratch test was detected in this group after 24 hr. In contrast tsEV group showed the highest invasion capacity after 24 hr.
  • Conclusion: Taken together, our results revealed that, tsEV modification for loading miR-34a might be considered as a new way of miR-34a delivery into triple negative breast cancer cells which may provide new insights toward using this method for miRNA-replacement therapy.
  • Keywords: Breast cancer, exosome, invasion, scratch test