Fig 1.
Dynamic regulation of collagen by the col1a1-miR-29b vector.
The col1a1 promoter works as a sensor for collagen expression that drives the expression of miR-29b, and therefore dynamically regulates collagen production and minimizes the off-target effect of miR-29b.
Fig 2.
Comparison of col1a1 and CMV promoter’s regulation of downstream gene expression.
(A) Both col1a1-luc and CMV-luc vectors were transfected into MEF cells for 24h. Luciferase activity was measured using a firefly luciferase substrate from Promega. (B) MEF cells transfected with col1a1-luc vector for 24 h were treated with TGF-β1 for an additional 24h and luciferase activity was measured. Each treatment was repeated 3 times (N = 3).
Fig 3.
Infection by lentiviral constructs in Mouse Embryonic Fibroblast (MEF) cells.
(A) The CMV-RFP/col1a1-EGFP vector was packed into lentivirus and mixed with polybrene. (B) MEF cells were cultured on a 6-well plate for 24h and infected with the lentiviruses for 48h. EGFP and RFP were visualized under a fluorescence microscope with a 20x lens.
Fig 4.
Infection of lentiviruses in Mouse Embryonic Fibroblast (MEF) cells in the lung tissue of mice.
The vector was packed into lentivirus and mixed with polybrene. About 100 μl of the mixture was delivered to the lung surface by the intra-trachea method. One week after the delivery, mice were euthanized and a frozen section of lung tissue was prepared. EGFP and RFP were visualized under a Leica confocal microscope.
Fig 5.
The effect of col1a1-miR-29b and CMV-miR-29b on collagen expression.
(A) Structure of Col1a1-miR-29b and CMV-miR-29b vectors. (B) Collagen expression. Col1a1-miR-29b and CMV-miR-29b vectors were transfected into MEF cells for 24h before treatment with TGF-β1 for an additional 24h. Total RNA was then extracted from the cell lysates. RT-qPCR was used to measure the expression of collagen. N = 7 for each group.
Fig 6.
The effect of col1a1-miR-29b and CMV-miR-29b on collagen synthesis.
The col1a1-miR-29b and CMV-miR-29b vectors were transfected into MEF cells for 24h before treatment with TGF-β1 for an additional 24h. Cell lysates (A) and culture medium (B) were collected for Western blot. Each of the above experiments were repeated 3 times (N = 3).
Fig 7.
Differentially Expressed Gene (DEG) profiling in MEF cells.
MEF cells transfected with empty vector, col1a1-miR-29b vector, or CMV-miR-29b vector, then treated with 20 ng/ml TGF-β for 24h. The total RNA was extracted for RNA-sequencing by LC Science. (A) The log2 foldchange of miR-29b expression cells in transfected cells. The log2 fold change and p-value were calculated using R-package DESeq2 as described in the Method section. (B) Volcano plot of significantly regulated genes after TGF-β treatment. (C) Heatmap of DEG in different groups.
Fig 8.
Representative gene map of upregulated KEGG pathways after TGF-β treatment.
Pathway analysis was performed using GAGE R-package and a gene map was obtained using Pathview R-package as described in the Method section.
Table 1.
TGF-β-induced KEGG pathway changes in MEF cells.
Table 2.
Downregulated KEGG pathways by Col1a1-miR-29b and CMV-miR-29b in MEF cells treated with TGF-β.
Fig 9.
miR-29b targeting gene expression profiling after miR-29b overexpression.
The FPKM value of gene expression was used for the heatmap. The top 100 miR-29b targeting gene lists were obtained from the miRDB database.