Fig. 4
The miRNA profiling and validation of short-term activated HSCs (3dHSCs) and long-term activated HSCs (14dHSCs) and corresponding sEVs. (A) Heatmap generated with DeSeq2 software packages showing the Euclidean distances between the samples. (B) Correlation of normalized reads (log2TPM) obtained by small RNA-Seq and adjusted RT-qPCR CT values for 15 miRNAs (rno-let-7f-5p, rno-miR-10a-5p, rno-miR-125a-5p, rno-miR-125b-1-3p, rno-miR-127-3p, rno-miR-139-5p, rno-miR-143-3p, rno-miR-146b-5p, rno-miR-27a-3p, rno-miR-27b-3p, rno-miR-30a-5p, rno-miR-30b-5p, rno-miR-423-5p, rno-miR-486, and rno-miR-99a-5p). RT-qPCR was performed with the same batch of RNAs prepared for sequencing. The relative expression levels of the selected miRNAs as determined by qPCR were expressed as adjusted Ct (ΔCt + 15) for cell samples and as (ΔCt + 25) for sEV samples. U6snRNA served as the reference gene for cell samples, and spike-in cel-miR-39 served as the reference gene for sEV samples. (C) Venn diagram showing the common and unique miRNAs identified in HSCs and HSC sEVs; the differentially expressed miRNAs between 3dHSCs and 14dHSCs and those between the corresponding sEVs were compared; only those miRNAs with a base mean > 100 were included (Table S6). The 12 miRNAs expressed differentially in either HSCs or the corresponding sEVs upon activation are listed on the right side; the up- or downregulation of these miRNAs is indicated by an upward red arrow or downward green arrow. (D) The expression of miR-99a-5p, miR-139-5p, and miR-221-3p in sEVs from culture-activated rat primary HSCs and the TGF-β-activated human hepatic stellate cell line LX2 was determined by RT-qPCR, and spike-in cel-miR-39 served as a reference. The relative expression of miRNA was expressed as adjusted Ct (40 - Ct), and statistical significance was determined by Student’s t test, *** p < 0.001, ** p < 0.01, * p < 0.05, ns, not significant. (E) Overall survival analyses of miR-99a-5p, miR-139-5p, and miR-221-3p expression in HCC patients based on TCGA survival data, and a 33rd percentile cutoff was adopted