Journal: Cell Death & Disease

Article Title: CircRNA TADA2A relieves idiopathic pulmonary fibrosis by inhibiting proliferation and activation of fibroblasts

doi: 10.1038/s41419-020-02747-9

Figure Lengend Snippet: a Circular RNAs (circRNAs) expression levels in IPF primary human lung fibroblasts (IPF-HLF; n = 5) and normal primary human lung fibroblasts (N-HLF; n = 5) were measured by qRT-PCR. * P < 0.05, ** P < 0.01 vs N-HLF. b Left: CircTADA2A expression in human IPF fibroblastic cell lines (LL-97A and LL-29) and normal human fibroblastic cell line (LL-24) was measured by qRT-PCR. Right: Representative fluorescence in situ hybridization (FISH) images of circTADA2A in LL-97A, LL-29, and LL-24 (blue, DAPI; red spot, positive staining; Scale bar = 20 µm). ** P < 0.01 vs LL-24 cells. c LL-24 cells were stimulated with fetal calf serum (FCS; 2% or 5%), platelet-derived growth factor-BB (PDGF-BB; 30 or 60 ng/ml), insulin-like growth factor 1 (IGF-1; 100 or 200 ng/ml), and transforming growth factor-β1 (TGF-β1; 5 or 10 ng/ml) for 6 h, respectively. CircTADA2A expression was measured by qRT-PCR. * P < 0.05, ** P < 0.01 vs LL-24 cells without stimulation.

Article Snippet: The human IPF fibroblastic cell lines (LL-97A and LL-29) and normal human fibroblastic cell line (LL-24) were obtained from the American Type Culture Collection (USA), authenticated and tested for mycoplasma contamination from the Procell (Chnia).

Techniques: Expressing, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Staining, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: miR-101 expression is down-regulated in fibrotic lungs. a, heat map of miRNA expression in IPF patients. Heat map of these dysregulated miRNAs was built by using GenePattern software (http://www.broadinstitute.org/cancer/software/genepattern).3 b, miR-101 expression in IPF lungs. miRNA microarray and real-time PCR were performed using total RNA isolated from the lung tissues of IPF patients. n = 10 for the <50% FVC group, n = 10 for the 50–80% FVC group, and n = 8 for the >80% predicted FVC group. Real-time PCR results were normalized to U6. Data were expressed as a ratio over the >80% FVC group. c and d, real-time PCR analysis showing the down-regulation of mature miR-101a, pri-miR-101a, and pri-miR-101b expression in the lungs (n = 6) and mature miR-101a expression in the fibroblasts (n = 3) isolated from saline (Sal)- and bleomycin (Bleo)-treated mice. The expression levels were normalized to U6 (for mature miR-101) or GAPDH (for pri-miR-101) and were expressed as a ratio of saline. The results are presented as the mean ± S.E. ANOVA followed by Tukey's HSD test was performed for multiple comparisons. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Cell culture HEK 293T cells and lung fibroblasts (IMR-90, HFL1, CCD-8Lu, and LL29) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Expressing, Software, Microarray, Real-time Polymerase Chain Reaction, Isolation, Saline

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Regulation of miR-101 expression by ETS. a, genomic structure of human miR-101-1 and the schematic presentation of potential ETS-binding sites in the upstream regions of the human miR-101-1 and miR-101-2 promoters. TSS, transcription start site. b and c, effects of ETS1/2 on human miR-101-1 and miR-101-2 promoter activities without or with ETS-binding site mutations in fibroblasts. Conserved ETS-binding site 1 (CCGGAAG) and site 5 (TTTCCTA) in human miR-101-1 promoter were mutated to site 1 (CCactgG) and site 5 (TTTagaA). Conserved ETS-binding site 2 (ATTCCTC) in human miR-101-2 promoter was mutated to site 2 (ATagcaC). HFL1 cells were transfected with 50 ng of the promoter reporter construct of miR-101-1 wild type (WT), miR-101-1-mute1, miR-101-1-mute5, miR-101-2 WT, and miR-101-2-mute2, 100 ng of the pCMV-ETS1, pCMV-ETS2 or control (CON) vector, and the normalization TK vector for 48 h. Firefly and Renilla luciferase activities were measured. ETS1 and ETS2 protein levels were determined by Western blotting. Data were expressed as a fold change over the control vector. n = 3. d and e, miR-101 expression in ETS1/2 knockdown CCD-8Lu fibroblasts. Fibroblasts were treated with lentiviral ETS1 or ETS2 shRNA or the virus control at an m.o.i. of 100 for 48 h. ETS1 and ETS2 protein levels were determined by Western blotting. miR-101 expression was determined by real-time PCR and normalized to U6 snRNA, n = 4. f, down-regulation of ETS1 and ETS2 mRNA expression in saline (Sal)- and bleomycin (Bleo)-treated mouse lung tissues. The expression of ETS1/2 was relative to GAPDH. n = 3. The results are presented as the mean ± S.E. ANOVA followed by Tukey's HSD test was performed for multiple comparisons. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Cell culture HEK 293T cells and lung fibroblasts (IMR-90, HFL1, CCD-8Lu, and LL29) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Expressing, Binding Assay, Transfection, Construct, Control, Plasmid Preparation, Luciferase, Western Blot, Knockdown, shRNA, Virus, Real-time Polymerase Chain Reaction, Saline

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: WNT5a increases fibroblast proliferation. a, real-time PCR analysis showing the high expression of WNT5a and WNT5b mRNAs in HFL1 and LL29 cells. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. b, WNT5a induces fibroblast proliferation. HFL1 and LL29 cells were serum-starved for 24 h and then stimulated for 12 h with 1 μg/ml WNT5a. Cell proliferation was determined using the BrdU cell proliferation kit. n = 3. c and d, knockdown of WNT5a suppresses fibroblast proliferation. HFL1 and LL29 cells were treated with a lentivirus expressing shRNA targeting WNT5a or the virus control (siCON) at an m.o.i. of 50 for 48 h. Cell proliferation was determined using a BrdU cell proliferation kit. n = 3. WNT5a protein expression was determined by Western blotting. The results are presented as the mean ± S.D. ***, p < 0.001.

Article Snippet: Cell culture HEK 293T cells and lung fibroblasts (IMR-90, HFL1, CCD-8Lu, and LL29) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Knockdown, shRNA, Virus, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: WNT5a induces fibroblast proliferation via WNT5a-FZD4/6-NFATc2 signaling. a, NFATc2 nuclear localization in fibroblasts induced by WNT5a. LL29 cells were serum-starved for 24 h and then stimulated with none (CON) or WNT5a (1 μg/ml) for 4 h. The cells were fixed for immunocytochemical analysis using anti-NFATc2 antibodies and Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained with DAPI. Scale bar, 50 μm. b, inhibition of fibroblast proliferation by CsA. LL29 cells were treated with CsA (1 μg/ml) for 24 h and then stimulated with 1 μg/ml WNT5a for 12 h. Cell proliferation was determined by the BrdU assay. n = 3. c and d, effects of silencing FZD4, FZD6, and NFATc2 on fibroblast proliferation. LL29 cells were infected with a lentivirus expressing shRNA targeting FZD4, FZD6, or NFATc2 (siFZD4, siFZD6, or siNFACTc2, respectively) or a virus control (siCON) at an m.o.i. of 50 for 48 h. Protein levels were determined by Western blotting. The silenced cells were serum-starved for 24 h and then stimulated with 1 μg/ml WNT5a for 12 h. Cell proliferation was determined by the BrdU assay. n = 3–6. e and f, WNT5a and NFATc2 expression in IPF patients. Real-time PCR were performed using total RNA isolated from the lung tissues of IPF patients. n = 10 for the <50% FVC group, n = 10 for the 50–80% FVC group, and n = 8 for the >80% predicted. The expression level was relative to GAPDH. The results are presented as the mean ± S.E. ANOVA followed by Tukey's HSD test was performed for multiple comparisons. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Cell culture HEK 293T cells and lung fibroblasts (IMR-90, HFL1, CCD-8Lu, and LL29) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Staining, Inhibition, BrdU Staining, Infection, Expressing, shRNA, Virus, Control, Western Blot, Real-time Polymerase Chain Reaction, Isolation

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: miR-101 inhibits WNT5a-induced fibroblast proliferation. a, effect of overexpression of miR-101 on fibroblast proliferation. LL29 cells were treated with a lentivirus expressing miR-101-1 or the virus control (VC) at an m.o.i. of 50 for 48 h. The cells were serum-starved for 24 h and then stimulated for 12 h with 1 μg/ml WNT5a. Cell proliferation was determined by the BrdU assay (n = 5). b, histological staining of BrdU showing the effect of WNT5a and miR-101 on fibroblast proliferation. Scale bar, 50 μm. c, nuclear amount of NFATc2 was decreased by the overexpression of miR-101. LL29 cells were treated with a lentivirus expressing miR-101-1 and then stimulated with WNT5a (1 μg/ml). Nuclei were extracted using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Waltham, MA) to determine the expression of the NFATc2 and H1 proteins by Western blotting. d, overexpression of miR-101-1 suppressed WNT5a-induced NFATc2 mRNA expression in LL29 cells by real-time PCR analysis. The expression levels were relative to GAPDH. n = 3. e, NFATc2 nuclear localization in the absence of FZD4 and FZD6. LL29 cells were split into a chamber slide at 2,000 cells per chamber. After a 24-h culture, cells were infected with lentiviral shFZD4 and shFZD6 (m.o.i. 25 for each) or shCon at m.o.i. = 50 for 48 h. Cells were serum-starved for another 24 h and stimulated with WNT5a (1 μg/ml) for 4 h. The cells were fixed for immunocytochemical analysis using anti-NFATc2 antibodies and Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained with DAPI. Scale bar, 50 μm. The results are represented as the mean ± S.E. ANOVA followed by Tukey's HSD test was performed for multiple comparisons. , p < 0.05; **, p < 0.01.

Article Snippet: Cell culture HEK 293T cells and lung fibroblasts (IMR-90, HFL1, CCD-8Lu, and LL29) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Over Expression, Expressing, Virus, Control, BrdU Staining, Staining, Extraction, Western Blot, Real-time Polymerase Chain Reaction, Infection

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: miR-101 controls α-SMA and collagen expression in lung fibroblasts. a and b, effect of miR-101 overexpression on TGF-β1-induced α-SMA and collagen protein and mRNA expression. LL29 cells were treated with lentiviral miR-101 or the virus control (vc) at an m.o.i. of 50 for 48 h. Then, the cells were stimulated with 5 ng/ml TGF-β1 for 48 h. Western blotting and real-time PCR were performed to determine the protein and mRNA expression levels. The expression levels were relative to GAPDH. n = 3 cell preparations. c and d, effect of miR-101 knockdown on the protein and mRNA expression of α-SMA, COL1A1, and COL3A1. CCD-8Lu fibroblasts were treated with lentiviral anti-miR-101 or the virus control at an m.o.i. of 50 for 72 h, and protein and mRNA expression was determined by Western blotting and real-time PCR. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. e, miR-101 suppresses TGFβ-1-induced collagen gel contraction in LL29 cells. f, LL29 cells were treated with lentiviral miR-101 or the virus control at an m.o.i. of 50 for 48 h. Then, the cells were stimulated with 5 ng/ml TGF-β1 for 48 h. The cells were fixed for immunocytochemical analysis with the anti-α-SMA antibody and Alexa Fluor 546-conjugated second antibody. Nuclei were stained with DAPI. Scale bar, 50 μm. The results are presented as the mean ± S.E. ANOVA followed by Tukey's HSD test was performed for multiple comparisons. *, p < 0.05; **, p < 0.01.

Article Snippet: Cell culture HEK 293T cells and lung fibroblasts (IMR-90, HFL1, CCD-8Lu, and LL29) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Expressing, Over Expression, Virus, Control, Western Blot, Real-time Polymerase Chain Reaction, Knockdown, Staining

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: miR-101 inhibits fibroblast activation through TGF-β-Smad2/3 signaling. a, ELISA analysis showing increased Smad2/3 phosphorylation by TGF-β1 treatment in LL29 cells. miR-101 suppressed the TGFβ1-induced Smad2/3 phosphorylation. The results are presented as the mean ± S.E. n = 4–6. *, p < 0.05. b, SMAD2/3 expression in IPF patients. Real-time PCR were performed using total RNA isolated from the lung tissues of IPF patients. n = 10 for the <50% FVC group, n = 10 for the 50–80% FVC group, and n = 8 for the >80% predicted. The expression level was relative to GAPDH.

Article Snippet: Cell culture HEK 293T cells and lung fibroblasts (IMR-90, HFL1, CCD-8Lu, and LL29) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Expressing, Real-time Polymerase Chain Reaction, Isolation

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Identification of FZD4, FZD6, and TGFBR1 as miR-101 targets. a, 3′-UTR reporter assay: FZD4-UTR-S1 (position 1–1,432) containing miR-101-binding site 1 (position 537–543), FZD4-UTR-S2 (position 4,178–5,463) containing miR-101-binding site 2 (position 1,240–1,246), full length of FZD6-UTR, TGFBR1-UTR-S1 (position 66–1,524) containing miR-101-binding site 1 (position 460–466), or TGFBR1-UTR-S2 (position 3,210–4,749) containing miR-101-binding site 2 (position 3,993–3,999) were co-transfected into HEK 293T cells with miR-101-1. The relative luciferase activities were measured using the Dual-Luciferase® reporter assay system. n = 3. b and c, effect of overexpressing or knockdown miR-101 on the protein expression of FZD4, FZD6, and TGFBR1 in fibroblasts. LL29 cells were treated with a lentivirus expressing miR-101-1 or the virus control (VC) at an m.o.i. of 50 for 48 h (b) or CCD-8Lu fibroblasts were treated with a lentivirus expressing anti-miR-101 or the virus control (Anti-CON) at an m.o.i. of 50 for 72 h. c, Western blotting was performed to determine the protein expression of FZD4, FZD6, and TGFBR1. The protein samples used here were the same as mentioned in Fig. 7c. The GAPDH immunoblot shown in Fig. 7c was used again here. d, overexpression of miR-101 inhibits FZD4, FZD6, and TGFBR1 mRNA expression in fibroblasts. LL29 cells were treated with lentiviral miR-101 or the virus control at an m.o.i. of 50 for 48 h. The mRNA expression of FZD4, FZD6, and TGFBR1 was determined by real-time PCR and normalized to GAPDH. The results are presented as the mean ± S.E. n = 3. e, real-time PCR showing the increased mRNA levels of FZD4, FZD6, and TGFBRR1 by anti-miR-101. CCD-8Lu fibroblasts were treated with lentiviral anti-miR-101 or the virus control at an m.o.i. of 50 for 72 h. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. f–h, mRNA expression of FZD4, FZD6, and TGFBR1 in IPF patient lungs. The mRNA levels of FZD4, FZD6, and TGFBR1 were determined by real-time PCR and normalized to β-actin. n = 8–10. The results are presented as the mean ± S.E. Statistical analyses were performed by using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Cell culture HEK 293T cells and lung fibroblasts (IMR-90, HFL1, CCD-8Lu, and LL29) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Reporter Assay, Binding Assay, Transfection, Luciferase, Knockdown, Expressing, Virus, Control, Western Blot, Over Expression, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Overexpression of FZD4, FZD6, or TGFBR1 rescues miR-101-mediated inhibition of fibroblast proliferation and activation. a–d, knockdown of TGFBR1 repressed the TGF-β1-induced mRNA expression of α-SMA, COL1A1, COL3A1, and COL4A1 in fibroblasts. LL29 cells were treated with lentivirus shRNA-TGFBR1 or the virus control at an m.o.i. of 50 for 48 h. Then, the cells were stimulated with 5 ng/ml TGF-β1 for 48 h. The α-SMA, COL1A1, COL3A1, and COL4A1 mRNA expression levels were determined by real-time PCR. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. The results are presented as the mean ± S.D. **, p < 0.01. e and f, overexpression of FZD4 or FZD6 rescues the miR-101-mediated inhibition of fibroblast proliferation. LL29 cells were split into 96-well plates at 3,000 cells per well. After a 24-h culture, cells were infected with lentiviruses at a total of m.o.i. = 50 (miR-Con miR-101, GFP, FZD4, and FZD6 at m.o.i. = 25 each, virus control (VC) = miR-Con + GFP) for 48 h. Cells were starved for another 24 h and stimulated with WNT5a (1 μg/ml) for 12 h. FZD4 and FZD6 expression was determined by Western blotting by using anti-GFP antibody. Cell proliferation was determined by BrdU assay. Data were presented as mean ± S.E. Statistical analysis was performed by ANOVA and followed by Tukey's HSD test. n = 4. **, p < 0.01. g–j, overexpression of TGFBR1 rescues miR-101-mediated inhibition of α-SMA, COL1A1, and COL3A1 mRNA expression. LL29 cells were infected with lentiviruses at a total of m.o.i. = 50 (miR-Con, miR-101, GFP, and TGFBR1 at m.o.i. = 25 each, virus control = miR-Con + GFP) for 48 h. Cells were then stimulated with TGFβ1 (5 ng/ml) for 48 h. Cells were collected for real-time PCR. The results are presented as the mean ± S.E., n = 3. ANOVA followed by Tukey's HSD test was performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Cell culture HEK 293T cells and lung fibroblasts (IMR-90, HFL1, CCD-8Lu, and LL29) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Over Expression, Inhibition, Activation Assay, Knockdown, Expressing, shRNA, Virus, Control, Real-time Polymerase Chain Reaction, Infection, Western Blot, BrdU Staining