Role of NRP1 in Bladder Cancer Pathogenesis and Progression

Figure 1. NRP1 is upregulated in BC. (A) NRP1 was overexpressed in BC tissues (n = 59) compared with that in normal bladder tissues (n = 59) in the Wen C et al. bladder dataset. (B) Expression of NRP1 was upregulated in muscle invasive BC tissues compared with that in non-muscle invasive BC tissue samples in the GSE3167, GSE65635, and GSE120736 datasets, respectively. (C) Representative IHC images of NRP1 in normal bladder tissues and in (D) BC tissues. (E) The levels of the NRP1 mRNA in SVHUC1 cell line and five BC cell lines examined using real-time PCR. The average NRP1 mRNA expression was normalised to the expression of β-actin. Three independent experiments were conducted for each assay and *p < 0.01 vs. the SVHUC1 group.

Figure 2. Downregulation of NRP1 reduces BC cells proliferation and angiogenesis. (A) T24 and 5637 cells were infected with lentivirus-expressing NRP1 shRNA-1, shRNA-2, and shRNA-3, or a control shRNA; the NRP1 mRNA level as measured using qRT-PCR. (B) Downregulation of NRP1 reduced the mean colony number in the colony formation assay. (C) MTT assays revealed that downregulation of NRP1 significantly reduced the growth rate of BC cells. (D) Downregulation of NRP1 reduced tubule formation of vascular endothelial cell. Three independent experiments were conducted for each assay, and data are presented as the mean ± standard error of the mean, *p < 0.01 vs. the control group.

Figure 3. NRP1 modulates BC cells apoptosis, cell cycle, migration, and invasion. (A) Apoptosis assay and quantitation of apoptotic cells of T24 and 5637 cells after NRP1 knockdown or control shRNA expression. (B) Flow cytometric analysis of T24 and 5637 cells following NRP1 knockdown or control shRNA expression. (C, D) Images and normalised migration (C) or invasion (D) of T24 and 5637 cells following NRP1 knockdown or control shRNA expression. Three independent experiments were conducted for each assay, and data are presented as the mean ± standard error of the mean, *p < 0.05 vs. the control group.

Figure 4. KEGG pathway analysis of DEGs following knockdown of NRP1. (A) Gene expression volcano plot of T24 cells transfected with NRP1 shRNA and a control shRNA vector. The red color on the left side represents 599 upregulated genes, and that on the right side represents 880 downregulated genes (log2FC> 2 and p-value < 0.05). (B) Heatmap and hierarchical cluster analysis of T24 cells transfected with NRP1 shRNA and a control shRNA vector. Column represents sample, and row represents gene, green represents a lower level gene expression, and red represents a relatively higher of gene expression. (C) Bubble plot of KEGG pathway analysis of upregulated DEGs. KEGG pathway description was assigned to y-axis and gene ratio was assigned to horizontal axis as the proportion of differential genes in the whole gene set. The dot size represents the gene counts in a certain pathway. (D) KEGG Chord plot of the relationship between the enrichment pathways and their corresponding genes in the upregulated DEGs list. A gene was linked to a certain pathway by the colored bands, and blue-to-red coding next to the genes indicates log FC. (E) KEGG Cluster of the upregulated DEG grouped by their functional…

Figure 5. The canonical pathway analysis by IPA®. (A) The enrichment of the DEGs in the canonical signaling pathway, sorted by –log(P). (B) The effect of experimental data on signal transfer in the interferon signaling pathway.

Figure 6. Validation of pathways associated with NRP1 silencing. (A) The gene network map of molecular mechanisms of cancer pathway including the potential NRP1-regulated genes. (B) The protein expression of some known tumor-associated genes in molecular mechanisms of cancer pathway were confirmed using western blot in T24 cells with NRP1 knockdown. Western blot was performed in three independent experiments and independently represent each internal control (GAPDH). (C) Western blot of ERK/MAPK related protein expression in T24 and 5637 BC cells after NRP1 knockdown. (D) Western blot of JNK/MAPK related protein expression in T24 and 5637 BC cells after NRP1 knockdown. (E) Proposed model for the molecular mechanisms underlying the action of NRP1 in BC progression. *p < 0.05 vs. the control group.

Figure 7. Disease and functional, upstream and network analysis by IPA®. (A) Disease and functional analysis using IPA® evaluated the positive or negative correlation between NRP1 and other diseases or functions, which were ranked by log (p-value). (B) The heatmap demonstrates the relationship between DEGs expression and activation or inhibition of diseases and functions categories. (C) Upstream analysis predicted that the NUPR1 regulatory network was activated after NRP1 was knocked down. (D) T24 and 5637 cells were infected with lentivirus-expressing NUPR1 or a control shRNA; the NUPR1 and NRP1 mRNA level was measured using qRT-PCR. (E) The molecular interaction network analysis predicted interaction among the molecules in the dataset and found that the top-ranked molecular interaction network was primarily enriched in the diseases and function categories of cancer, organismal injury and abnormalities and cell cycle, which including the altered genes after NRP1 silencing are shown. (F) The regulatory effect network analysis revealed that NRP1 may be a regulator in neovascularisation activation. (G) the mRNA expression of FOXP3 and FGF2 gene in 5637 cells with NRP1 silencing were measured…

Figure 8. Co-expressed genes analysis and ceRNA network prediction. (A) NRP1 mRNA expression in various cancer cell lines obtained from CCLE database. The abscissa is the tumor type and sample size, and the ordinate is the expression of target genes. (B) NRP1 mRNA expression in 26 urinary tract cancer cells extracted from CCLE database. We took the logarithm of the original data for better visualization. (C) Heatmap of the top 20 co-expressed genes with NRP1 in urinary tract cancer cells in both upregulated and downregulated groups. (D) the mRNA expression of DCBLD2 in 5637 cells with NRP1 silencing were measured using qRT-PCR. *p < 0.05 vs. the control group. (E) The significantly enriched signal transduction pathways of co-expressed genes of NRP1 in urinary tract cancer cells obtained using GSEA analysis. (F) A ceRNA sankey diagram of NRP1 constructed using the data extracted from TCGA database.