Fig 1: Epigenetic changes in the MMP2 gene promoter and globally in human bladder cells expressing ANGA. Graphical analysis of CpG methylation pattern in the MMP2 promoter gene. The percentages of methylated CpGs in UROtsaANG, UROtsaEmpty, RT112KD-ANG and RT112Scr cells were noted. *p < 0.01. B. Hierarchical cluster analysis of methylation data profiling in UROtsaANG, UROtsaEmpty and RT112KD-ANG and RT112Scr cells. Each row represents a methylated gene and each column a sample. The scale represents standard deviations from the mean after a Z-transformation of signal values of a gene across all samples. Red represents a higher level of gene expression and green a lower level, relative to the mean across all samples for each gene. C. Illumina chip analysis of the changed DNA methylation patterns of MMP2. Hypermethylation and hypomethylation: the status of MMP2 DNA methylation in UROtsaANG compared to UROtsaEmpty, and RT112KD-ANG compared to RT112SCR. D. Pathway analysis of the identified genes with changed promoter DNA methylation status. Eleven of the most significantly enriched pathways were plotted for genes of hypermethylation (left panel) or hypomethylation (right panel) in UROtsaANG compared to UROtsaEmpty. E. Methylation status of 48 tumor suppressor genes within the Illumina whole genome methylation array in UROtsaANG compared to UROtsaEmpty.
Fig 2: ANG overexpression in human bladder cell lines leads to MMP2 expressionA. ANG and MMP2 levels were evaluated in UROtsa, RT4, RT112, 5637, UM-UC-3, T24, TCCSUP, and UM-UC-14 by immunoblotting. B. UROtsa cells were stably transfected with empty vector (UROtsaEmpty) or ANG expression plasmid (UROtsaANG), while RT112 cells were transiently transfected with negative control siRNA (RT112Scr) or siRNA directed at ANG mRNA (RT112KD-ANG). The mRNA levels were analyzed by quantitative RT-PCR and protein levels were analyzed by immunoblotting analysis. ß-Actin was used as an internal loading control. C. ANG ELISA assay also confirmed altered secretion of ANG in the same cell lines. Zymogen assays were performed to assess the activity of MMP2. Three independent experiments were performed in triplicate. *p < 0.05; vs. UROtsaEmpty, and ˆp < 0.05; vs. RT112Scr.
Fig 3: A hypothetical representation of the regulatory pathway underlying ANG-induced MMP2 expressionBased on this study, ANG has two different effects on the cells a) induces DNMT3a expression, b) represses DNMT3b expression. Reduced DNMT3b levels lead to hypomethylation of the MMP2 promoter, which induces MMP2 gene transcription. Thus, expression of MMP2 contributes to increase invasive potential of bladder cancer and reduced disease free survival.
Fig 4: High ANG levels are associated with low DNMT3b and high MMP2 levels, which are related to poor prognosis in human bladder tumorsA. Analysis of ANG, DNMT3b and MMP2 mRNA levels and methylation status (percentages of methylated CpG islands) of MMP2 gene in muscle invasive bladder cancer and non-muscle invasive bladder cancer. B. Kaplan-Meier survival rate analysis for ANG, DNMT3b and MMP2 expression levels and methylation status of MMP2 gene in human bladder cancer. Tumor samples with patient history (see Table 1) were used for survival analysis of A-D.
Fig 5: DNMT3b and MMP2 are highly regulated by ANG overexpressionA. mRNA and protein expression levels of DNMT1, DNMT3a and DNMT3b were assessed in UROtsaANG, UROtsaEmpty, RT112KD-ANG and RT112Scr cells. *p < 0.05; vs. UROtsaEmpty, and ˆp < 0.05; vs. RT112Scr. ß-actin was used as an internal control. B. The effects of DNMT3a and DNMT3b knockdown on MMP2 mRNA and protein levels were assessed. The mRNA levels were analyzed by qRT-PCR and protein levels were analyzed by immunoblotting analysis. ß-actin was used as an internal control. C. The effects of DNMT3a and DNMT3b knockdown on percentages of methylated CpG islands of the MMP2 promoter were assessed using Epitect methyl II PCR array. Depicted is one of three independent experiments, which were performed in triplicate. *p < 0.05; vs. Empty-Scr or Scr-Scr, and ˆp < 0.05; vs. ANG-Scr or KD-ANG - Scr.
Supplier Page from Abcam for Human Angiogenin ELISA Kit (ANG)