Journal: Molecular Therapy. Nucleic Acids

Article Title: Exosomal Transmission of MicroRNA from HCV Replicating Cells Stimulates Transdifferentiation in Hepatic Stellate Cells

doi: 10.1016/j.omtn.2019.01.006

Figure Lengend Snippet: Effects of HCV Replication on Exosome Release, Exosomal RNA Level, and HSC Activation (A) Comparison of exosome release from naive Huh-7 (Huh-7 exo) and JFH-1 stable cells (JFH-1 exo). Immunoblot analysis of exosome marker proteins (CD63, LAMP2, HSP70, and CD81) in increasing volume of exosomes purified from each cell type is shown (left). The level of each exosome marker protein was quantified and presented as a percentage of that in Huh-7 exo (middle). Exosome total proteins were quantified using a bicinchoninic acid assay (BCA) (right). (B) Immunoblot analysis of total cell lysates (lysate) and isolated exosomes (exo) of naive Huh-7 or JFH-1 stable cells is shown. (C and D) Quantification of miRNAs and HCV RNA within exosomes from naive Huh-7 or JFH-1 cells is shown. After isolating exosomes, we re-suspended the exosomes in 100 μL PBS buffer and used 2 μL or 20 μL of the solution for the analysis of (C) the RNA levels of miR-192 (left: Huh-7 exo, 3.03 ± 2.69 × 10 5 copies per 2 μL and 3.09 ± 2.26 × 10 6 copies per 20 μL; JFH-1 exo, 7.20 ± 4.74 × 10 5 copies per 2 μL and 8.80 ± 7.89 × 10 6 copies per 20 μL), miR-122 (right: Huh-7 exo, 2.25 ± 1.80 × 10 5 copies per 2 μL and 1.60 ± 1.43 × 10 6 copies per 20 μL; JFH-1 exo, 4.43 ± 2.29 × 10 5 copies per 2 μL and 5.06 ± 4.90 × 10 6 copies per 20 μL), and (D) HCV RNA (JFH-1 exo, 2.42 ± 3.07 × 10 5 copies per 2 μL and 2.15 ± 2.79 × 10 6 copies per 20 μL). (E and F) Quantification of fibrotic marker RNAs and proteins in LX-2 cells treated with each type of exosomes is shown. (E) Relative intracellular mRNA levels of fibrosis markers (left) and copy numbers of miR-192 (right) in LX-2 cells treated with each type of exosomes are shown. Copy numbers of miR-192 were 3.30 ± 1.67 × 10 5 and 6.18 ± 2.78 × 10 5 per 100 ng of total RNA in Huh-7 exo-treated and JFH-1 exo-treated LX-2 cells, respectively. (F) Immunoblot analysis of the fibrosis markers COL1A1 and α-SMA, as well as TGF-β1, in LX-2 cells treated with each type of exosome (left) and quantification of their levels relative to those of tubulin (right) are shown. (G) Effects of exosomes on HSC activation are shown. LX-2 cells were treated with each exosome type for 72 h, fixed, stained, and visualized using fluorescence microscopy. Representative images (left) of α-SMA and DAPI staining in LX-2 cells are shown. Scale bars represent 50 μm. α-SMA protein levels (right) in LX-2 cells treated with each type of exosomes were quantified from more than five fields of three independent experiments and shown relative to the levels in cells treated with Huh-7 exo. Data are also shown in Figure S4 . All data are presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent the SEM. p values were determined using a one-tailed unpaired Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Proteins quantified using the SMART bicinchoninic acid Protein Assay (iNtRON Biotechnology, Gyeonggi-do, Republic of Korea) were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore) in 2 mM Tris-192 mM glycine buffer.

Techniques: Activation Assay, Comparison, Western Blot, Marker, Purification, Acid Assay, Isolation, Staining, Fluorescence, Microscopy, One-tailed Test