Table 1.
ChIP-Seq and RNA-Seq datasets with corresponding ID.
Fig 1.
Correlation plot for high grade PDAC ChIP-Seq dataset and peak signal intensity.
(A) Spearman correlation plot for low and high grade PDAC ChIP-Seq dataset. Correlation comparison of peak signal intensity for (B) Low grade PDAC cell lines and (C) High grade PDAC cell lines.
Fig 2.
Analysis of high grade PDAC dataset for differential expressed genes using Cytoscape.
(A) Representative network cluster for biological process that are actively involved in cell differentiation and structural development were identified using BinGO. Nodes represented in white were not significantly expressed, while a deeper shade of orange correlated with processes predicted with confidence. The inset represents a cluster of biological processes with p-value ≤ 0.05. These clusters were involved in cell differentiation and structural formation. (B) Gene interaction network obtained using GeneMANIA was conducted on selected genes identified from important biological processes. Black nodes represent the selected genes, while grey nodes were automatically drawn by the tool based on their connectivity to the highlighted ones. All edges in the network were derived from prior knowledge.
Fig 3.
Pathway analysis using ClueGO indicated that several identified genes were interconnected to cancer related pathways.
(A) Representation of KEGG pathways enriched in PDAC gene set (Bonferroni step-down corrected p ≤ 0.05). Pathways in cancer (highlighted in a red box) was found to be interconnected to other over-represented terms (B) Pie-chart of pathways expressed in PDAC for gene set extracted from pathways in cancer, with essential pathways marked in red.
Fig 4.
Predicted gene topological relation as obtained from HGPEC.
Fig 5.
RNAi based silencing of PAX2 in MIA PaCa-2 and PANC-1 cells led to an increase in drug susceptibility.
(A) MIA PaCa-2 and (B) PANC-1 cells were transfected using indicated siRNAs for 48hours. Cell viability assay was performed post 48 hours using luminometer. Percent cell viability was calculated with respect to Negative control siRNA (siNeg). siCell Death, an siRNA used as Positive cell death phenotype control, from qiagen, USA was used as a positive control. Experiments were done independently in triplicates SEM = ± 3. The viability of (C) MIA PaCa-2 and (D) PANC-1 cell lines, previously transfected with siNeg control or siPAX2 siRNA and subsequently (24 hours post transfection) exposed to increasing concentrations of chemotherapeutic drugs camptothecin or 5’Flurouracil for 48 hr (mean ± SEM, n = 3) at each drug concentration normalized to vehicle (V, 0.1% DMSO). (E) Caspase 3/7 activity in MIA PaCa-2 (top panel) or PANC-1 (bottom panel) cell lines, previously transfected with siNeg control or siPAX2 siRNA and subsequently (24 hours post transfection) exposed to indicated concentrations of chemotherapeutic drugs camptothecin or 5’Flurouracil (5’FU) for 48 hours (mean ± SEM, n = 3) at each drug concentration normalized to vehicle (V, 0.1% DMSO). Relative fold change in caspase activity was measured using caspase 3/7 Glo assay using luminometer. (F) The viability of MIA PaCa-2 parental (Pa-MIA PaCa-2) or Gemcitabine resistance MIA PaCa-2 (Res-MIA PaCa-2) cell lines, previously transfected as indicated, with siNeg control or siPAX2 siRNA and subsequently (24 hours post transfection) exposed to increasing concentrations of Gemcitabine for 48 hours (mean ± SEM, n = 3) at each drug concentration normalized to vehicle (V, 0.1% DMSO).
Fig 6.
Effect of PAX2 on the transcriptional regulation of ABC family of transporters and survival analysis.
(A) MIA PaCa-2 cells were transfected with siRNAs against PAX2. Relative mRNA levels were analysed with respect to relative mRNA levels (siNEG) 48hours post transfection. Normalization was performed with 18sRNA levels. Experiments were done independently in triplicates SEM = ± 3. (B) MIA PaCa-2 cells were transfected with siNeg or siPAX2 siRNAs. ChIP assay was then performed with PAX2 antibody or control IgG antibody. The fold enrichment of coprecipitating DNA was determined by qPCR for the indicated promoters. Error bars are means ± SD of three independent experiments with triplicate samples. Survival analysis was performed using Surv Express software [72]. Patient datasets used (C) [73], (D) [74], [75], (E) TCGA pancreatic adenocarcinoma, (F) ICGC portal, for the multivariant analysis of indicated genes ABCC2, ABCC5, ABCC3, ABCC1 and PAX2. High (blue) and low (yellow) represents gene expression.