Figure 5: Sorting of miR-193a from cell to exosomes through major vault protein (MVP).

(a) Biotin-miR-193a complex was pulled down from whole cell extracts using streptavidin beads and then analysed by electrophoresis followed by Coomassie blue staining (left panel). MALDI-TOF analysis of tryptic peptides (right panel) from the band indicated (left panel). (b) Western blot analysis expression of MVP proteins from before (top panel) and after streptavidin pulldown (bottom panel) of lysates of CT26 cells transfected with Bio-miR-193a or control miRNA. (c) MVP knockout (KO) CT26 cells were generated using the CRISPR/Cas9 system. qPCR-quantification of mature miR-193a, MVP, Caprin1, CyclinD and c-MYC expressed in CT26 cells (left panel) and CT26 exosomes (right panel) after the cells were treated as indicated. (d) Western blot analysis showing the level of MVP, Caprin1, CCND2 and c-MYC in cell lysates treated as indicated. (e) Proliferation of MVP KO CT26 cells treated as indicated. Cell viability was detected from day 0 to 5 after transfection. (f) Schematic representation (left panel) of treatment schedule as indicated. Representative livers (middle top panel) (metastatic nodules shown by arrows) and H&E-stained sections of livers (middle bottom panel, × 400 magnification, scale bar 200 μm) from tumour-bearing BALB/c mice (n=5 per group). Liver weight (right, top panel) and number of metastatic foci in liver (right bottom panel) were quantitatively analysed. (g) Mature miR-193a in tumour tissue (left panel) and tumour exosomes (middle panel) was quantified by qPCR. Survival analysis of BALB/c mice after intrasplenic injection of CT26 cells treated as indicated (right panel) (n=9 per group). (h) Representative images of xenografts in SW620 tumour-bearing nude mice (left panel) (n=5 mice per group). Changes of tumour volumes in an SW620 xenograft model (right panel). Liver tumour volume was used to evaluate tumour size using the following formula: nodule volume=(width)² × length/2. (i) qPCR-quantification of mature miR-193a in exosomes and tissues of tumour in SW620 xenograft mice. *P<0.05 (two-tailed t-test); NS represents non-significance. Each data point was measured in triplicate (error bars, s.e.m.).