Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Unveiling the role of microRNA-7 in linking TGF-β-Smad-mediated epithelial-mesenchymal transition with negative regulation of trophoblast invasion.

doi: 10.1096/fj.201801898RR

Figure Lengend Snippet: Figure 1. TGFBR1 regulated cell invasion and interacted with EMT-related TFs in trophoblasts. A, B) Lentiviral transduction of shTGFBR1 efficiently down-regulated the expression of TGFBR1 (A) and its protein level (B) in both 3A-sub E and HTR-8/SVneo trophoblast cell lines. The quantification of TGFBR1 expression relative to GAPDH is shown (n = 3) (A). C) TGFBR1 silencing enhanced trophoblast invasion in Matrigel-coated Transwell assay. D) Quantitative results of invasion assay and Western blot analysis are shown (n = 6). E, F) Supplementation of TGF-b1 and transduction of shTGFBR1 significantly activated SNAIL and SLUG expression in 3A-sub E (E) and HTR-8/SVneo cells (F). However, the expression of TWIST was not altered by the administration of TGF-b1 (n = 4). Data information: 1-way ANOVA. Quantitative data are presented as means 6 SD (A, B, D–F), and statistically significant differences between groups are indicated with asterisks. ****P # 0.0001.

Article Snippet: TGFBR1 overexpression vectors, including humanwild-type (WT) TGFBR1 (pCMV5B-TGFBR1-His) and human mutant dominant negative (KR) TGFBR1 [pCMV5B-TGFBR1 (K-R)-HA], were gifts from Joan Massagué (Memorial Sloan Kettering Cancer Center, New York City, NY, USA) (plasmids 19161 and 19159; Addgene,Watertown, MA, USA).

Techniques: Transduction, Expressing, Transwell Assay, Invasion Assay, Western Blot

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Unveiling the role of microRNA-7 in linking TGF-β-Smad-mediated epithelial-mesenchymal transition with negative regulation of trophoblast invasion.

doi: 10.1096/fj.201801898RR

Figure Lengend Snippet: Figure 2. The differential effect of normal and malignant trophoblasts in response to silencing TGFBR1 involves miR-7. A) Heat map of miR expression in shTGFBR1-silenced trophoblasts compared with the SC. Up-regulation and down-regulation of miR expression compared with the scrambled-transduced cells are shown in red and green, respectively. miR-7 is one of the down- regulated miRs in cells with shTGFBR1 compared with the SC. B, C) TGRBR1 silencing, either via antagonizing by the selective TbRI inhibitor (SB431542) or shTGFBR1 transduction, resulted in significant reduction in miR-7 expression in both cell lines. 3A- sub E control/SB431542: n = 3; HTR-8/SVneo control: n = 5; HTR-8/SVneo SB431542: n = 4 (B). 3A-sub E SC: n = 6; 3A-sub E shTGFBR1#1: n = 5; 3A-sub E shTGFBR1#2: n = 6; HTR-8/SVneo SC/shTGFBR1#1/shTGFBR1#2: n = 3 (C). D) Constitutive expression of TGFBR1 by transduction in TD mutant resulted in significantly high miR-7 expression compared with KR mutant and WT (n = 5). E) TGRBR1 silencing by shTGFBR1 transduction resulted in significant induction of miR-7 expression in both JAR and JEG-3 cell lines (JAR SC, shTGFBR1#1, and shTGFBR1#2: n = 8, 5, and 5; JEG-3 SC, shTGFBR1#1, and shTGFBR1#2: n = 10, 4, and 7). EV, empty vector. Data information: all of the above experiments were performed without TGF-b1 supplementation; 1- way ANOVA for data, which are presented as means 6 SD (B–E). Statistically significant differences between groups are indicated with asterisks. *P , 0.05, **P , 0.01, ****P # 0.0001.

Article Snippet: TGFBR1 overexpression vectors, including humanwild-type (WT) TGFBR1 (pCMV5B-TGFBR1-His) and human mutant dominant negative (KR) TGFBR1 [pCMV5B-TGFBR1 (K-R)-HA], were gifts from Joan Massagué (Memorial Sloan Kettering Cancer Center, New York City, NY, USA) (plasmids 19161 and 19159; Addgene,Watertown, MA, USA).

Techniques: Expressing, Transduction, Control, Mutagenesis, Plasmid Preparation

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Unveiling the role of microRNA-7 in linking TGF-β-Smad-mediated epithelial-mesenchymal transition with negative regulation of trophoblast invasion.

doi: 10.1096/fj.201801898RR

Figure Lengend Snippet: Figure 4. miR-7 regulated trophoblast invasion through EMT. A–F) TGFBR1 silencing (A–C) and miR-7 knockdown (D–F) significantly increased the expression of EMT-related TFs, including SNAIL, SLUG, and TWIST. 3A-sub E SC, shTGFBR1#1, and shTGFBR1#2: n = 5, 3, and 3; HTR-8/SVneo SC, shTGFBR1#1, and shTGFBR1#2: n = 5, 4, and 4 (A). 3A-sub E SC, shTGFBR1#1, and shTGFBR1#2: n = 5, 2, and 4; HTR-8/SVneo SC, shTGFBR1#1, and shTGFBR1#2: n = 5, 4, and 3 (B). 3A-sub E SC, shTGFBR1#1, shTGFBR1#2: n = 6, 4, and 3; HTR-8/SVneo SC, shTGFBR1#1, and shTGFBR1#2: n = 6, 3, and 3 (C). 3A-sub E SC, shTGFBR1#1, and shTGFBR1#2: n = 5, 4, and 3; HTR-8/SVneo SC, shTGFBR1#1, and shTGFBR1#2: n = 5, 4, and 5 (D). 3A-sub E SC, shTGFBR1#1, and shTGFBR1#2: n = 4, 4, and 3; HTR-8/SVneo SC, shTGFBR1#1, and shTGFBR1#2: n = 5, 5, and 4 (E). 3A-sub E SC, shTGFBR1#1, and shTGFBR1#2: n = 6, 3, and 4; HTR-8/SVneo SC, shTGFBR1#1, and shTGFBR1#2: n = 6, 5, and 6.) (F). G, H) TGFBR1 silencing increased the level of EMT-related TFs and their downstream targets (N-cadherin, VE-cadherin, vimentin, and MMP-9). The level of the epithelial marker E-cadherin was reduced in shTGFBR1 transduced cells compared with the SC. However, the level of (continued on next page)

Article Snippet: TGFBR1 overexpression vectors, including humanwild-type (WT) TGFBR1 (pCMV5B-TGFBR1-His) and human mutant dominant negative (KR) TGFBR1 [pCMV5B-TGFBR1 (K-R)-HA], were gifts from Joan Massagué (Memorial Sloan Kettering Cancer Center, New York City, NY, USA) (plasmids 19161 and 19159; Addgene,Watertown, MA, USA).

Techniques: Knockdown, Expressing, Marker

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Unveiling the role of microRNA-7 in linking TGF-β-Smad-mediated epithelial-mesenchymal transition with negative regulation of trophoblast invasion.

doi: 10.1096/fj.201801898RR

Figure Lengend Snippet: Figure 5. TGFBR1 regulated miR-7 expression via Smad2-dependent pathway. A) Transduction of TGFBR1 overexpression construct (TD) demonstrated an increase in TGFBR1 and pSmad2 expression compared with KR mutants and WT and induced a down-regulation of EMT TFs (SNAIL and TWIST) and their downstream effectors (N-cadherin, vimentin, VE-cadherin, and MMP-9) and up-regulation of E-cadherin compared with WT and KR mutants, indicating an impairment of EMT. B) Transduction of aOn-TGFBR1-TD plasmid in 3A-sub E cells treated with Dox resulted in forced expressions of TGFBR1 and (continued on next page)

Article Snippet: TGFBR1 overexpression vectors, including humanwild-type (WT) TGFBR1 (pCMV5B-TGFBR1-His) and human mutant dominant negative (KR) TGFBR1 [pCMV5B-TGFBR1 (K-R)-HA], were gifts from Joan Massagué (Memorial Sloan Kettering Cancer Center, New York City, NY, USA) (plasmids 19161 and 19159; Addgene,Watertown, MA, USA).

Techniques: Expressing, Transduction, Over Expression, Construct, Plasmid Preparation

Journal: European Journal of Human Genetics

Article Title: Distinct variants affecting differential splicing of TGFBR1 exon 5 cause either Loeys–Dietz syndrome or multiple self-healing squamous epithelioma

doi: 10.1038/s41431-018-0127-1

Figure Lengend Snippet: Analysis of TGF-β signaling by dual luciferase assay. (a) Western blot analysis for TGFBR1 expressions from generated constructs. (b, c) SBE-firefly luciferase activity in HEK293T cells. (b) HEK293T cells transfected with the indicated constructs were cultured for 36 h, and after 4 h starvation with FCS-free DMEM, the cells were exposed to recombinant human TGF-β1 (Wako) at a concentration of 5 ng/mL for 8 h. n = 3, ***p < 0.001 versus the untreated-control (Student’s t-test). (c) HEK293T cells were transfected with the indicated constructs and harvested for 48 h after transfection. n = 3, ***p < 0.001, *p < 0.05 (Analysis of Variance test)

Article Snippet: HEK293T cells were co-transfected with TGFBR1 expression vectors, SMAD3:SMAD4 inducible firefly luciferase reporter plasmid (pGL4.48 (luc2P/SBE/Hygro), Promega) and constitutively active renilla luciferase plasmid (pRL-SV40, Promega) using Lipofectamine 2000.

Techniques: Luciferase, Western Blot, Generated, Construct, Activity Assay, Transfection, Cell Culture, Recombinant, Concentration Assay, Control

Journal: European Journal of Human Genetics

Article Title: Distinct variants affecting differential splicing of TGFBR1 exon 5 cause either Loeys–Dietz syndrome or multiple self-healing squamous epithelioma

doi: 10.1038/s41431-018-0127-1

Figure Lengend Snippet: TGFBR1 variant analysis. (a) Genomic DNA sequencing revealed a heterozygous single-base substitution (c.973 + 1 G > A) in the proband. (b–d) Splice analysis of exon 5. (b) Arrows indicate the position of PCR (top panel). Wild-type and variant cDNA were predicted to produce 400 bp and 232 bp fragments. The actual PCR products on 1.2% agarose gel (bottom panel) in a healthy unaffected volunteer (left, WT) and the proband (right). The doublet bands in the gel indicate the splicing abnormality. (c, d) Direct sequencing of cDNA from wild-type (top) and variant (bottom) alleles in the proband revealed a 168 bp deletion corresponding to entire exon 5 skipping (c) and a last 9 bp deletion of exon 5 (d). aa amino acid position of TGFBR1

Article Snippet: HEK293T cells were co-transfected with TGFBR1 expression vectors, SMAD3:SMAD4 inducible firefly luciferase reporter plasmid (pGL4.48 (luc2P/SBE/Hygro), Promega) and constitutively active renilla luciferase plasmid (pRL-SV40, Promega) using Lipofectamine 2000.

Techniques: Variant Assay, DNA Sequencing, Agarose Gel Electrophoresis, Sequencing

Journal: European Journal of Human Genetics

Article Title: Distinct variants affecting differential splicing of TGFBR1 exon 5 cause either Loeys–Dietz syndrome or multiple self-healing squamous epithelioma

doi: 10.1038/s41431-018-0127-1

Figure Lengend Snippet: The ex vivo splicing assay. (a) Natural and putative splice sites in the region around TGFBR1 exon 5 with S&S splice site scores. The putative splicing donor site 9 bp upstream of the 5′ natural splice site and splice acceptor site 76 bp downstream from the 3′ natural splice site were predicted to contribute to the pathogenesis of LDS and MSSE, respectively. (b) The pTBNde(min) minigene system used to analyze the effect of TGFBR1 variants on ex vivo pre-mRNA splicing. (c) Splicing products amplified by RT–PCR using 2–3α and B2 primers shown in (b) were separated by 1.2% agarose gel electrophoresis. The identity of the spliced products was established by direct sequencing (d–f) and is schematically represented on the right. (d–f) Direct sequencing of cDNA from wild-type (top) and variant (bottom) alleles. In the small and large products of LDS variant alleles, the entire exon 5 (168 bp) (d) and last 9 bases of exon 5 (e) were missing, respectively. (f) For the MSSE variant allele, the first 76 bp bases of exon 5 was missing. FN-EDB, fibronectin-extra domain B; aa, amino acid position of TGFBR1

Article Snippet: HEK293T cells were co-transfected with TGFBR1 expression vectors, SMAD3:SMAD4 inducible firefly luciferase reporter plasmid (pGL4.48 (luc2P/SBE/Hygro), Promega) and constitutively active renilla luciferase plasmid (pRL-SV40, Promega) using Lipofectamine 2000.

Techniques: Ex Vivo, Splicing Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing, Variant 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: 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: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

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: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression, Inhibition, Activation Assay, Knockdown, Expressing, shRNA, Virus, Control, Real-time Polymerase Chain Reaction, Infection, Western Blot, BrdU 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: Adenovirus-mediated miR-101 gene transfer attenuates bleomycin-induced pulmonary fibrosis. On day 1, an adenovirus expressing miR-101-1 or the control adenovirus (VC) (5 × 109 pfu per mouse) was delivered into the lungs of 6–8-week-old C57BL/6 mice by nasal instillation. On day 2, bleomycin (Bleo) (0.06 units per mouse) or the same volume of saline (Sal) was intranasally instilled into the lungs. On day 15, the mice were subjected to the analysis of respiratory mechanics by Flexivent and then sacrificed. The left lungs were collected for RNA and protein analysis, and the right lungs were fixed for histological analysis. a, real-time PCR analysis showing adenovirus-mediated overexpression of miR-101 in the mouse lung. The expression levels were relative to U6. b, H&E staining showing the fibrotic changes in the mouse lung induced by bleomycin. miR-101 attenuated the fibrotic changes in bleomycin-treated mouse lungs. Scale bar, 100 μm. c, Ashcroft score grade of pulmonary fibrosis. d, lung collagen content as determined using the QuickZyme hydroxyproline assay kit. e and f, real-time PCR analysis showing the increased COL1A1 and COL3A1 mRNA levels in bleomycin-treated mouse lungs and miR-101 suppression of the increase of COL1A1 and COL3A1 mRNA levels. The expression levels were relative to GAPDH. g and h, analysis of lung mechanics by Flexivent analysis. Elastance (Ers) was measured in a single-compartment model. h was measured in the Constant-Phase model. i and j, NFATc2, FZD4, FZD6, and TGFBR1 mRNA expression was increased in bleomycin-treated mouse lungs, and the increase was suppressed by miR-101 treatment as determined by real-time PCR analysis. The expression level was relative to GAPDH. n = 5 for saline + VC and saline + miR-101; n = 7–10 for bleomycin + VC and bleomycin + miR-101. 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: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Expressing, Control, Saline, Real-time Polymerase Chain Reaction, Over Expression, Staining, Hydroxyproline 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: Primers for the construction of plasmids FW is forward, and RE is reverse.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression