Basic research of pancreatic cancer cell-derived extracellular vesicles

Objective To isolate extracellular vesicles (VEs) from the culture supernatant of pancreatic cancer (PANC-1) and human pancreatic ductal epithelial cells (HPDE6-C7), and to compare the differential expression of microRNA (miRNA, miR)-483-5p in the two kinds of cells and their secreted EVs. Methods Ultracentrifugation method was used to prepare the complete culture medium of depletion of serum-derived vesicles. Cell count Kit (CCK-8) was used to detect the effect of complete medium on cell proliferation without ultracentrifugation (control group) and with ultracentrifugation (experimental group). Cells were cultured in vesicle-depleted complete medium, and EVs were extracted by ultracentrifugation. The concentration of EVs was detected by BCA protein concentration determination kit and nanoparticles tracking analysis (NTA). Transmission electron microscope, NTA, and Western blotting were used to identify whether they, obtained by ultracentrifugation, were in accordance with the characteristics of EVs. The expression of miR-483-5p was detected by quantitative real time polymerase chain reaction (qPCR). The results of CCK-8 proliferation test and qPCR test were expressed by mean±standard deviation (SD). The statistical method was t test. Results The results of CCK-8 proliferation test showed that there was no significant difference between the experimental group and the control group after 48 h and 72 h in HPDE6-C7 cells (HPDE6-C7, 0.674±0.036 vs. 0.671±0.016, t=0.315, P48 h>0.05; 0.890±0.027 vs. 0.925±0.099, t=0.581, P72 h>0.05), as well as the PANC-1 (0.759±0.004 vs. 0.761±0.016, t=0.249, P48 h>0.05; 1.114±0.025 vs. 1.145±0.014, t=1.898, P72 h>0.05). The transmission electron microscopy showed that the EVs were "tea-cup tray" . The results of NTA showed that the diameter of 98% PANC-1 derived EVs was 130.0 nm, and that of 98% HPDE6-C7 derived EVs was 129.7 nm. Western blotting showed that CD63 and TSG101 were positive, but GM130 was negative. The concentration of EVs secreted by PANC-1 and HPDE6-C7 was measured by protein concentration assay kit and NTA as follows: 1.5 g/L, 1.510 11 particles/mland 1.3 g/L, 1.610 11 particles/ml, respectively. Compared with HPDE6-C7, the expression of miR-483-5p in PANC-1 was significantly increased (2.820±0.180 vs. 1.000±0.006, t=-17.539, P<0.01). Compared with HPDE6-C7 derived EVs, the expression of miR-483-5p in PANC-1 derived EVs was significantly increased (3.503±0.265 vs. 1.002±0.084, t=-15.582, P<0.01). Conclusion The ultracentrifugation method does not affect the normal proliferation of cells while removing fetal bovine serum-derived vesicles in the complete medium; the identification results show that the isolated pellets correspond to the characteristics of EVs. According to the results of NTA, it can be judged that they should be classified as small EVs. The results of qPCR show that miR-483-5p is highly expressed in PANC-1 and the secreted EVs. Key words: Pancreatic cancer; Extracellular vesicles; Ultracentrifugation; MicroRNA-483-5p