Figure 2. MiRNAs Are the Functional Extracellular Vesicle Contents That Aggravate Colorectal Cancer Growth in Fatty Liver.

(A) A heatmap of miRNAs. MiRNA PCR array for (1) EVs from the sera of healthy controls and patients with NAFLD, (2) EVs from mouse sera (8-week-LFD or HFD feeding), (3) EVs from sera of metastatic tumor-bearing mice fed a LFD or HFD, (4) EVs from primary hepatocytes (PHCs) of LFD or HFD-fed mice, and (5) EVs from vehicle (Veh)-treated or PA-treated mouse PHCs. The heatmaps illustrate the log2 (fold change) values. The heatmap diagram highlights six common miRNAs. miR-103, miR-25, and miR-92a are the top three differentially expressed miRNAs.

(B) Effect of miR-25, miR-92, and miR-103 on colony formation. Representative images from the colony-forming assay of MC38 cells(upper), and the average colony numbers per field(lower) (n=4/group).

(C) Transwell migration and invasion assay. MC38 cells were transfected with 50 nM control, miR-25, miR-92, or miR-103 mimics. Representative pictures are shown(upper). Quantification of migrated and invaded cells(lower) (n=4/group).

(D,E) Effect of EVs with compound inhibition of miR-25, miR-92, and miR-103. Mouse PHCs were transfected with a combination of three antagomiRs or a control (100 nM each) for 48 hours. Then, cells were treated with 400 μM PA for an additional 24 hours. EVs were collected from the cells transfected with antagomiRs (PA-EV^miR-i) or a control (PA-EV^Control).

(D) Colony-forming assay. MC38 cells were treated with PA-EV^miR-i or PA-EV^Control (n=4/group).

(E) Transwell migration and invasion assay. MC38 cells were treated with PA-EV^miR-i or PA-EV^Control for 48 hours and then placed in the top chamber, and the migration and invasion to the lower chamber were quantified (n=4/group).

Data shown as mean ± SEM (B-E). Significance determined by one-way analysis of variance (ANOVA) with Tukey’s post hoc analysis (B,C) or two-tailed Student’s t-test (D,E).

See also Figure S2 and Table S1.