Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P < 0.001) destruction of the network created by the HUVECs following the treatment with LY3039478 (1 µM). The concomitant administration of MMP13 counteracts significantly ( P < 0.01) drug effectiveness.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P < 0.001 calculated with Student’s t test. b NOTCH1 gene and its pro-angiogenic targets are overexpressed in human intrahepatic cholangiocarcinoma (iCCA). Levels of NOTCH1, DLL4, VEGFA, and MMP13 mRNA were significantly more elevated in iCCA ( n = 42) than corresponding nontumorous surrounding livers (SL; n = 42), as detected by quantitative reverse-transcription PCR. Number target (NT) = 2 −ΔCt , wherein ΔCt value of each sample was calculated by subtracting the average Ct value of the gene of interest from the average Ct value of the β-actin gene. Mann–Whitney test: vs SL, P < 0.0001. c Expression of the NOTCH1 gene correlates with mRNA levels of putative target genes (HES1, DLL4, VEGFA, and MMP13) in a collection of human intrahepatic cholangiocarcinoma (CCA) samples ( n = 42). Linear regression analysis was used. d Representative expression patterns of CK19, NOTCH1, HES1, DDL4, and MMP13 in human intrahepatic cholangiocarcinoma (iCCA) as detected by immunohistochemistry. Upper panels: CCA case (CCA1) showing strong, concomitant immunoreactivity for NOTCH1, HES1, DDL4, and MMP13. Lower panels: CCA specimens (CCA2) exhibiting low levels of NOTCH1, HES1, DDL4, and MMP13. As expected, both iCCA display robust immunolabeling for CK19 (a biliary marker). Magnification: ×200; scale bar = 100 μm. H&E hematoxylin and eosin staining.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing, Reverse Transcription, MANN-WHITNEY, Immunohistochemistry, Immunolabeling, Marker, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing

Journal: PLoS ONE

Article Title: A Feedback Inhibition between miRNA-127 and TGFβ/c-Jun Cascade in HCC Cell Migration via MMP13

doi: 10.1371/journal.pone.0065256

Figure Lengend Snippet: ( A ) Sequence alignment between miR-127 and the 3′UTR of MMP13 in human, mouse, and rat. Solid line: seed match region. ( B ) Transient transfection assay. Hela cells were co-transfected with the control pTarget (0), pTarget-miR-127, and MMP13 (left), PPP1R9B (middle), or MMP13 mutant (mut) 3′UTRs (right). Luciferase (Luc) activity was normalized to Renilla activity (act). PPP1R9B served as a negative control. ( C ) Transient transfection assay to determine the effect of pTarget-miR-127 (100 ng) and miR-127 inhibitor (20 nM) on MMP13 3′UTR reporter activity. ( D ) Western blots (WB) of MMP13 protein expression in MHCC97H cells that were transfected with pTarget-miR127 (2 µg) or treated with a miR-127 inhibitor (anti-miR-127) (20 nM). ( E ) qPCR analysis of miR-127 expression under the same experimental conditions as ( D ). ( B-E): Data are expressed as means ± SD of triplicate assays. *p<0.01, miR-127 vs. pTarget control; ¥ p<0.01, anti-miR-127 vs. negative control.

Article Snippet: Recombinant MMP13 protein was purchased from ProSpec-Tany TechnoGene Ltd. MMP13 inhibitor was purchased from Santa Cruz.

Techniques: Sequencing, Transient Transfection Assay, Transfection, Control, Mutagenesis, Luciferase, Activity Assay, Negative Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: A Feedback Inhibition between miRNA-127 and TGFβ/c-Jun Cascade in HCC Cell Migration via MMP13

doi: 10.1371/journal.pone.0065256

Figure Lengend Snippet: ( A ) Cell migration assay. MHCC97H cells were transfected with MMP13 siRNA (siMMP13) (20 nM) in the absence or presence of pTarget or pTarget-miR-127 (2 µg). After cells were seeded onto the inserts, lower chamber media were treated with MMP13 peptide (50 ng/ml) as indicated. Cells that had migrated through the membrane were fixed and stained with crystal violet (left). Quantitative results on the right. ( B-C ) qPCR analysis of MMP13 ( B ) and miR-127 ( C ) expression under the same experimental conditions as ( A ). ( D ) Cell migration assay. MHCC97H and Hepa-1 cells were transfected with pTarget or pTarget-miR-127 (2 µg), in the absence or presence of TGFβ (5 ng/ml). Migrated cells were stained with crystal violet and visualized by microscopy (left). Quantitative results on the right. ( A-D ): *p<0.01, miR-127 vs. pTarget in control group; ¥ p<0.01, pTarget in MMP13 group vs. pTarget in control group; ‡ p<0.01, miR-127 vs. pTarget in MMP13 group; § p<0.01, miR-127 vs. pTarget in siMMP13 group.

Article Snippet: Recombinant MMP13 protein was purchased from ProSpec-Tany TechnoGene Ltd. MMP13 inhibitor was purchased from Santa Cruz.

Techniques: Cell Migration Assay, Transfection, Membrane, Staining, Expressing, Microscopy, Control

Journal: PLoS ONE

Article Title: A Feedback Inhibition between miRNA-127 and TGFβ/c-Jun Cascade in HCC Cell Migration via MMP13

doi: 10.1371/journal.pone.0065256

Figure Lengend Snippet: ( A ) qPCR analysis of MMP13 (left), c-Jun (middle), and miR-127 (right) expression in MHCC97H cells treated with TGFβ. *p<0.01, TGFβ (+) vs. TGFβ (-) group. ( B ) Transient transfection assay. Hela cells were co-transfected with miR-127 promoter (pro) luciferase (luc) reporter, and c-Jun or c-Fos expression plasmids. *p<0.01, c-Jun (+) vs. c-Jun (-) group. ( C) Transient transfection assay. Hela cells were co-transfected with miR-127 or AP1 promoter reporter along with c-Jun/c-Fos plasmid (100 ng) in the absence or presence of TGFβ (5 ng/ml). *p<0.01, c-Jun/Fos vs. control without (-) or with (+) TGFβ; ¥ p<0.01, control with (+) TGFβ vs. control without (-) TGFβ. ( D ) Transient transfection assay. Hela cells were co-transfected with miR-127 promoter reporter, c-Jun (100 ng) and/or p53 plasmids (100 ng). *p<0.01, c-Jun or p53 vs. control; ¥ p<0.01, c-Jun/p53 vs. p53. ( B-D ) Luciferase (Luc) activity was normalized by Renilla activity (act). ( E ) qPCR analysis of miR-127 and p53 expression in HepG2 cells transfected with control (con) or p53 siRNAs (si-p53) (20 nM), or in HCT116 p53 +/+ (+) and HCT116 p53 −/− (-) cells. *p<0.01, si-p53 vs. control. ( F ) qPCR analysis of miR-127 expression in MHCC97H cells that were overexpressed with c-Jun and/or p53. *p<0.01, p53 vs. control (-); ¥ p<0.01, c-Jun/p53 vs. p53.

Article Snippet: Recombinant MMP13 protein was purchased from ProSpec-Tany TechnoGene Ltd. MMP13 inhibitor was purchased from Santa Cruz.

Techniques: Expressing, Transient Transfection Assay, Transfection, Luciferase, Plasmid Preparation, Control, Activity Assay

Journal: PLoS ONE

Article Title: A Feedback Inhibition between miRNA-127 and TGFβ/c-Jun Cascade in HCC Cell Migration via MMP13

doi: 10.1371/journal.pone.0065256

Figure Lengend Snippet: ( A-C ) qPCR analysis of c-Jun ( A ), miR-127 ( B ), and MMP13 ( C ) mRNA expression in MHCC97H cells treated with TGFβR inhibitor (SB 431542, 10 µM), NFκB inhibitor (BAY-11-7082, 5 µM, ERK inhibitor (PD 98059, 50 µM), p38 inhibitor (SB 203580, 10 µM) and JNK inhibitor (SP 600125, 50 µM),) in the absence (−) or presence of TGFβ (+) (5 ng/ml). Statistical results represent the mean ± SD. *p<0.01, TGFβ (+) vs. TGFβ (−) group.

Article Snippet: Recombinant MMP13 protein was purchased from ProSpec-Tany TechnoGene Ltd. MMP13 inhibitor was purchased from Santa Cruz.

Techniques: Expressing

Journal: PLoS ONE

Article Title: A Feedback Inhibition between miRNA-127 and TGFβ/c-Jun Cascade in HCC Cell Migration via MMP13

doi: 10.1371/journal.pone.0065256

Figure Lengend Snippet: ( A-B ) qPCR analysis of miR-127 expression ( A ) and MMP13 mRNA ( B , left), and Western blot (WB) analysis of MMP13 protein ( B , right) in 5 pairs of surrounding controls and HCC specimens. *p<0.01, tumor vs. non-tumor.

Article Snippet: Recombinant MMP13 protein was purchased from ProSpec-Tany TechnoGene Ltd. MMP13 inhibitor was purchased from Santa Cruz.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: A Feedback Inhibition between miRNA-127 and TGFβ/c-Jun Cascade in HCC Cell Migration via MMP13

doi: 10.1371/journal.pone.0065256

Figure Lengend Snippet: TGFβ activates the oncogene c-Jun through ERK and JNK pathways. The activation of c-Jun serves a dual function, which involves induction of MMP13 gene expression but repression of miR-127 gene transcription by inhibiting miR-127 promoter activity. c-Jun also antagonizes p53 activation of the miR-127 promoter and gene transcription. On the other hand, overexpression of miR-127 decreases MMP13 protein levels by binding to its 3′UTR and causing MMP13 degradation, thus diminishing TGFβ-mediated HCC migration.

Article Snippet: Recombinant MMP13 protein was purchased from ProSpec-Tany TechnoGene Ltd. MMP13 inhibitor was purchased from Santa Cruz.

Techniques: Activation Assay, Gene Expression, Activity Assay, Over Expression, Binding Assay, Migration