Volume 24, Issue 10, Pages 1848-1859 (October 2016) MiR-29a Assists in Preventing the Activation of Human Stellate Cells and Promotes Recovery From Liver Fibrosis in Mice  Yoshinari Matsumoto, Saori Itami, Masahiko Kuroda, Katsutoshi Yoshizato, Norifumi Kawada, Yoshiki Murakami  Molecular Therapy  Volume 24, Issue 10, Pages 1848-1859 (October 2016) DOI: 10.1038/mt.2016.127 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Preparation of two chronic liver injury models. (a,g) Treatment schedules for carbon tetrachloride (CCl4) and thioacetamide (TAA). (b,h) Hematoxylin and eosin (upper rows) and sirius red (SR) staining (lower rows) of liver. Scale bar = 200 μm. (c,i) SR staining positive area of liver. (d,j) Liver hydroxyproline levels. (e,k) The expression pattern of four fibrosis-related genes (COL1A1, α-SMA, MMP2, and TIMP2) in liver. (f,l) The expression pattern of miR-29a in liver. *P < 0.05 in Tukey HSD. Horizontal axis in (c–f) and (i–l) shows whether CCl4 treatment was done or not (CCl4) and the timing of sacrifice (observation). Molecular Therapy 2016 24, 1848-1859DOI: (10.1038/mt.2016.127) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Therapeutic effect of miR-29a in carbon tetrachloride (CCl4)-treated mouse liver for 1 and 2 weeks. (a) Summary of schedule for miRNA treatment. (b,c) Hematoxylin and eosin (upper row) and sirius red (SR) (lower row) staining in livers. Scale bar = 200 μm. (d) SR staining positive area of livers. (e) Liver hydroxyproline levels. (f) The expression pattern of COL1A1 in livers. *P < 0.05 in Tukey HSD, **P < 0.05 in student's t-test. Molecular Therapy 2016 24, 1848-1859DOI: (10.1038/mt.2016.127) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Therapeutic effect of miR-29a in thioacetamide (TAA)-treated mouse liver for 1 and 2 weeks. (a,b) Hematoxylin and eosin (upper row) and sirius red (SR) (lower row) staining in livers. Scale bar = 200 μm. (c) SR staining positive area of livers. (d) Liver hydroxyproline levels. (e) The expression pattern of COL1A1 in livers. *P < 0.05 in Tukey HSD, **P < 0.05 in student's t-test. Molecular Therapy 2016 24, 1848-1859DOI: (10.1038/mt.2016.127) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Microarray analysis of carbon tetrachloride (CCl4) and thioacetamide (TAA) liver fibrotic models and miR-29a target genes. (a) Venn diagram of the fibrosis-related genes selection criteria in six patterns. Number depicted in common genes in two or four groups. Black circle shows CCl4 1 week, dotted circle shows TAA 1 week, bold black circle shows CCl4 2 weeks, and bold dotted circle shows TAA 2 weeks. (b) MiR-29a target site sequences in 3′ UTR of PDGFC, FGL2, MAP4K4, and COL1A1. Numbers left of 5′-UTR sequences show nucleotide from 5′ end. MAP4K4 and COL1A1 have pleural recognition site of miR-29a. (c) The expression pattern of three target genes from microarray data. (d) The expression pattern of three target genes from real-time PCR data. PDGFC shows four sample's data (CCl4 1 week/2 weeks and TAA 1 week/2 weeks). Fgl2 shows two sample's data (CCl4/TAA 1 week). Map4k4 shows two sample's data (CCl4/TAA 2 weeks). *P < 0.05 in student's t-test. Molecular Therapy 2016 24, 1848-1859DOI: (10.1038/mt.2016.127) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Pharmacodynamics of miR-29a in mice. (a) The expression patterns of miR-29a in liver 3, 6, 24, and 48 hours after injecting miR-29a without treatment with any fibrotic inducing reagents. (b) The expression pattern of miR-29a in 1 week/2 weeks therapeutic carbon tetrachloride (CCl4) models. (c) The expression pattern of miR-29a in 1 week/2 weeks therapeutic thioacetamide (TAA) models. (d) The expression pattern of miR-29a in co-immunoprecipitated miRNA with Ago2 in 1-week therapeutic CCl4 model liver. White bars show NC miRNA-treated liver and black bars show miR-29a-treated liver. *P < 0.0001 in student's t-test. Molecular Therapy 2016 24, 1848-1859DOI: (10.1038/mt.2016.127) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 In vitro analysis of miR-29a function. (a) Time course of experiment for LX-2 cells. (b) The expression level of miR-29a in LX-2 treated with TGFβ1. (c) The expression level of fibrosis-related genes in LX-2. (d) Western blotting of COL1A1 and β-actin in LX-2 cells. (e) Cell viability test in LX-2 measured by XTT assay. (f) Apoptosis test in LX-2 verified by Caspase 9 activity. (c,e,f) White bars show NC miRNA transfected LX-2 and black bars show miR-29a transfected LX-2. *P < 0.05 in student's t-test. Molecular Therapy 2016 24, 1848-1859DOI: (10.1038/mt.2016.127) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Inhibitory effect of TGFβ activation in LX-2 cells. (a) Time course of experiment. MiR-29a was transfected to LX-2 cells; the medium was changed after 6 hours and TGFβ1 was added. The cells were isolated 24 and 48 hours after transfection. (b) The expression of fibrosis-related genes in LX-2 cells. White bars show NC miRNA transfected LX-2 and black bars show miR-29a transfected LX-2. (c) Western blotting of COL1A1 and β-actin in LX-2 cells on 2 days. *P < 0.05 in Tukey HSD, **P < 0.05 in student's t-test. Molecular Therapy 2016 24, 1848-1859DOI: (10.1038/mt.2016.127) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Schema of miR-29a for liver fibrosis. The summary of hypothetical effect of miR-29a on liver fibrosis in this study. The graphs show microarray data. White bars show NC miRNA-treated liver and black bars show miR-29a-treated liver. (a) MiR-29a can control COL1A1, PDGFC, FGL2, and MAP4K4 as target genes. The expression of four PDGFC pathway genes, RhoA, MAPK8, RAF1, and SRF, were downregulated in the miR-29a-treated group. (b) IL-1B, NDRG1, and BCL2 were indirectly controlled by miR-29a and expression pattern of these genes was observed in microarray. Molecular Therapy 2016 24, 1848-1859DOI: (10.1038/mt.2016.127) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions