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. 2019 Oct 4;11(19):8484-8501.
doi: 10.18632/aging.102334. Epub 2019 Oct 4.

CircUBAP2-mediated competing endogenous RNA network modulates tumorigenesis in pancreatic adenocarcinoma

Rongjie Zhao  1 , Junjie Ni  2 , Si Lu  3 , Sujing Jiang  4 , Liangkun You  1 , Hao Liu  1 , Jiawei Shou  1 , Chongya Zhai  1 , Wei Zhang  1 , Shengpeng Shao  5 , Xinmei Yang  6 , Hongming Pan  1 , Weidong Han  1
Affiliations

CircUBAP2-mediated competing endogenous RNA network modulates tumorigenesis in pancreatic adenocarcinoma

Rongjie Zhao et al. Aging (Albany NY). .

Abstract

We investigated the role of the competing endogenous RNA (ceRNA) network in the development and progression of pancreatic adenocarcinoma (PAAD). We analyzed the expression profiles of PAAD and normal pancreatic tissues from multiple GEO databases and identified 457 differentially expressed circular RNAs (DEcircRNAs), 19 microRNAs (DEmiRNAs) and 1993 mRNAs (DEmRNAs). We constructed a ceRNA network consisting of 4 DEcircRNAs, 3 DEmiRNAs and 149 DEmRNAs that regulates the NF-kappa B, PI3K-Akt, and Wnt signaling pathways. We then identified and validated five hub genes, CXCR4, HIF1A, ZEB1, SDC1 and TWIST1, which are overexpressed in PAAD tissues. The expression of CXCR4, HIF1A, ZEB1, and SDC1 in PAAD was regulated by circ-UBAP2 and hsa-miR-494. The expression of CXCR4 and ZEB1 correlated with the levels of M2 macrophages, T-regulatory cells (Tregs) and exhausted T cells in the PAAD tissues. The expression of CXCR4 and ZEB1 positively correlated with the expression of CTLA-4 and PD-1. This suggests that CXCR4 and ZEB1 proteins inhibit antigen presentation and promote immune escape mechanisms in PAAD cells. In summary, our data suggest that the circUBAP2-mediated ceRNA network modulates PAAD by regulating the infiltration and function of immune cells.

Keywords: ceRNA; circRNA; immune microenvironment; pancreatic adenocarcinoma.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1
Differentially expressed circRNAs, miRNAs and mRNAs in pancreatic adenocarcinomas (PAAD). Volcano plots and heatmaps show identification of (A) DEcircRNAs in GSE69362, (B) DEcircRNAs in GSE79634, (C) DEmiRNAs in GSE60980 (GPL15159) and (D) DEmRNAs in GSE60980 (GPL14550) between PAAD tissues and adjacent normal pancreatic tissue. The red color indicates upregulated genes in the PAAD tissues and blue color indicates downregulated genes, while black color indicates genes with no significant differences between the PAAD and normal pancreatic tissues. Heatmaps show the expression patterns of DEcircRNAs, DEmiRNAs and DEmRNAs. The PAAD and adjacent normal pancreatic tissues are represented by red and blue color, respectively. (E) Venn diagrams show commonly upregulated DEcircRNAs and downregulated DEcircRNAs in the PAAD tissues in both GSE69362 and GSE79634. Purple and orange circles indicate the number of DEcircRNAs in the GSE69362 and GSE79634 datasets, respectively. The red circles in the middle indicate the overlapping circRNAs between the two datasets. (F) Venn diagram shows the intersection between DEmiRNA-predicted targets obtained from miRDB, miRTarBase and TargetScan databases and DEmRNAs in GSE60980 (GPL14550).
Figure 2
Figure 2
The circRNA-miRNA-mRNA interaction network in the PAAD tissues. The circRNA-miRNA-mRNA interaction network consists of 4 circRNAs (hsa_circ_0007367, hsa_circ_0003958, hsa_circ_0043278 and hsa-circ-0049783), 3 miRNAs (hsa-mir-324-3p, hsa-miR-214 and hsa-miR-494), and 149 mRNAs. The diamond nodes indicate the circRNAs; rectangle nodes indicate the miRNAs; the elliptical nodes indicate the mRNAs. The edges indicate a possible connection between the circRNAs, miRNAs, and the mRNAs. The red and blue color indicates high and low expression of the ceRNAs in the PAAD tissues, respectively.
Figure 3
Figure 3
Basic structural features of the 4 PAAD-specific circRNAs. The structural features of (A) hsa_circRNA_0003958, (B) hsa_circRNA_0007367, (C) hsa_circRNA_0043278, and (D) hsa_circRNA_0049783 downloaded from the Cancer-Specific CircRNA Database (CSCD) are shown. The microRNA response element (MRE) is shown in red; The RNA binding protein (RBP) is shown in blue; The open reading frame (ORF) is shown in green.
Figure 4
Figure 4
Sankey plots of signaling pathways and biological processes related to differentially expressed mRNAs in PAAD tissues. Sankey plots show (A) signaling pathways and (B) biological processes (BP) related to DEmRNAs involved in PAAD based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and Gene Ontology_Biological Process (GO_BP) analyses, respectively.
Figure 5
Figure 5
Protein-protein intersection (PPI) network analyses of the DEmRNAs involved in the ceRNA network. The PPI network consists of 141 edges and 50 nodes representing proteins and interactions, respectively. The relative thickness of the edges represents the degree of relationship (weak, moderate, or strong) between the nodes.
Figure 6
Figure 6
Comparative analyses of the transcription levels of hub genes in GSE60980 (GPL14550), and related miRNAs in GSE60980 (GPL15159) and circRNAs in GSE79634 between PAAD and normal pancreatic tissues. (A–K) Transcription levels of circ-HIBADH, circ-UBAP2, circ-TADA2A, circ-CLEC17A, hsa-miR-214, hsa-mir-324-3p, hsa-miR-494, CXCR4, HIF1A, SDC1, TWIST1 and ZEB1 between PAAD (red) and normal pancreatic tissues (blue).
Figure 7
Figure 7
Validation of the transcript (mRNA) and protein levels of the hub genes using the Gene Expression Profiling Interactive Analysis (GEPIA) and The Human Protein Atlas database. (A) The transcript (mRNA) of the hub genes, namely, CXCR4, HIF1A, SDC1, TWIST1, and ZEB1 in PAAD and normal pancreatic tissues are shown. (B) Immunohistochemical (IHC) staining data of hub genes as follows: HIF1A protein expression in a PAAD tumor tissue (Staining: low; Intensity: weak; Quantity: 75%-25%; Location: cytoplasmic/membrane); HIF1A protein expression in a representative normal pancreatic tissue (Staining: low; Intensity: weak; Quantity: 75%-25%; Location: nuclear); SDC1 protein expression in a PAAD tumor tissue (Staining: medium; Intensity: moderate; Quantity: 75%-25%; Location: cytoplasmic/membrane). SDC1 protein expression in a representative normal pancreatic tissue (Staining: low; Intensity: moderate; Quantity: < 25%; Location: cytoplasmic/membrane). ZEB1 protein expression in a PAAD tumor tissue (Staining: low; Intensity: weak; Quantity: > 75%; Location: cytoplasmic/membrane). ZEB1 protein expression in a representative normal pancreatic tissue (Staining: low; Intensity: moderate; Quantity: < 25%; Location: cytoplasmic/membrane). The database lacked information regarding CXCR4 and TWIST1 protein expression in PAAD tumor tissues and normal pancreatic tissues. IHC results consistent with changed trend of transcript (mRNA) of hub genes in GSE60980 (GPL14550) and GEPIA were displayed.
Figure 8
Figure 8
The survival curves of hub genes and their related miRNAs based on Kaplan Meier. (A–D) Transcription levels of hsa-miR-324, hsa-miR-494, SDC1 and TWIST1 are significantly related to the overall survival of patients with PAAD (P < 0.05). Red curve indicates high expression in PAAD tumor tissues; black curve indicates low expression in PAAD tumor tissues; P value < 0.05 indicates statistical significance; HR: hazard ratio.
Figure 9
Figure 9
The two hub genes positively correlate with macrophage polarization in PAAD tissues. (A–D) The relationship between CXCR4 expression and biomarkers of monocytes, TAMs, M1 and M2 macrophages is shown. (E–H) The relationship between ZEB1 expression and biomarkers of monocytes, TAMs, M1 and M2 macrophages is shown. Note: Monocyte markers: CD86 and CSF1R; TAM markers: CCL2, CD68 and IL10; M1 macrophage markers: NOS2, IRF5 and PTGS2; M2 macrophage markers: CD163, VSIG4 and MS4A4A. Hub genes are shown on the x-axis, and the corresponding marker genes are shown on the y-axis.
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