Synthesis of exomiR-34a

Exosomes were purified from the medium of HEK293 cells as described by Thery et al with little modification.19 Briefly, HEK293 cells were cultured in the logarithmic growth phase, and then cells were seeded in ten 75 cm² flasks; each flask with 25 mL culture medium. After the cells were grown to 60–70% abundance, the original medium was removed and replaced with the same amount of fresh exosome-free serum medium. After the cell growth reached about 80–95% abundance, the supernatant was collected by centrifugation at 10,000 × g for 30 min at 4°C to remove the residual debris, and then the supernatant was filtrated through 0.22 μM polyvinylidene difluoride filters. The filtrated solution was centrifuged at 100,000 × g for 70 min at 4°C and the exosomes pellets thus obtained were washed twice with phosphate-buffered saline (PBS) and resuspended in 50 μL PBS. Butyleyanoacrylate (BCA) protein assay kit (Thermo Scientific, CA, USA) was used to determine the total protein content of exosomes. The size distribution and zeta potential of the isolated exosomes were measured by Nano-Zetasizer 90 at 37°C as per the manufacturer’s instructions.20 The morphology of exosomes after negative staining was observed by transmission electron microscopy.21 Western blot analysis was performed to determine the expression of the characteristic proteins of exosomes including CD81, CD63, CD9, etc.22

In order to analyze the distribution and the concentration of miRNAs, the miRNAs were labeled cy3 at the 5ʹ end of the miRNAs. The exomiR-34a was synthesized using an ultrasound approach. Briefly, the exosomes isolated above were dissolved in PBS solution, and miR-34a (mass ratio of miRNA: exosomes = 1:5) was added to the PBS solution and ultrasonicated at 20 W, for 5 s, stopping for 2 s and then the solution was allowed to stand on ice for 20 min. The loading rate of miRNA in exosomes was measured by agarose gel electrophoresis, stained with GelRed (Vazyme, China), and analyzed by agel-imaging system (BioRad, USA).