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. 2020 Jun 23;10:1013. doi: 10.3389/fonc.2020.01013

Figure 6.

Figure 6

Intercellular miR-622 transfer by EVs during EMT and the phenotype of PDAC cells. Panc-1 cells (1 × 106) were plated in 10 ml of vesicle-depleted medium on 10-cm dishes and were transfected with 12.5 nM miR-622 or the control mimic. After incubation for 72 h, EVs were extracted from the conditioned medium by ultracentrifugation. (A) Transmission electron microscopy was performed on EVs isolated from Panc-1 cells. Obtained EVs were composed of a homogeneous population of particles. (B) EV RNA was isolated and miR-622 or HULC expression was assessed by qRT-PCR. (C–F) EVs were added to recipient Panc-1 cells. (C) After 48 h, RNA was extracted from recipient cells and expression of miR-622, HULC, E-cadherin, N-cadherin, vimentin, and Snail was examined by qRT-PCR. RNA expression was normalized to that of U6 and expressed relative to the value of the control. (D) After 72 h, protein was extracted, and western blotting for E-cadherin, N-adherin, vimentin, and Snail was performed. Protein levels were normalized to that of β-actin. (E) After 24 h, cell invasion was examined by Transwell assay using an inverted microscope. (F) After 24 h, cell migration was examined by wound healing assay. Reference indicates the image at time 0. Bars are the means ± SEM of three independent experiments. *P < 0.05.