The Urinary Bladder Transcriptome and Proteome Defined by Transcriptomics and Antibody-Based Profiling

Abstract

To understand functions and diseases of urinary bladder, it is important to define its molecular constituents and their roles in urinary bladder biology. Here, we performed genome-wide deep RNA sequencing analysis of human urinary bladder samples and identified genes up-regulated in the urinary bladder by comparing the transcriptome data to those of all other major human tissue types. 90 protein-coding genes were elevated in the urinary bladder, either with enhanced expression uniquely in the urinary bladder or elevated expression together with at least one other tissue (group enriched). We further examined the localization of these proteins by immunohistochemistry and tissue microarrays and 20 of these 90 proteins were localized to the whole urothelium with a majority not yet described in the context of the urinary bladder. Four additional proteins were found specifically in the umbrella cells (Uroplakin 1a, 2, 3a, and 3b), and three in the intermediate/basal cells (KRT17, PCP4L1 and ATP1A4). 61 of the 90 elevated genes have not been previously described in the context of urinary bladder and the corresponding proteins are interesting targets for more in-depth studies. In summary, an integrated omics approach using transcriptomics and antibody-based profiling has been used to define a comprehensive list of proteins elevated in the urinary bladder.

Fig 1. Classification of all protein-coding genes based on their expression in human urinary bladder tissue.

(A) Scatterplot showing the FPKM values of all 20,344 protein-coding genes in the two urinary bladder samples, with each gene colored according to category. (B) Pie chart showing the distribution of all protein-coding genes into five categories based on transcript abundance and number of detected tissues, including expression in all 32 tissues (blue), mixed expression with genes expressed in a varying number of tissue (green), genes with elevated expression in urinary bladder (pink), not detected in urinary bladder (light grey), and not detected in any tissue (dark grey). The genes with elevated expression in urinary bladder are further subdivided depending on the degree of specificity as tissue enriched genes, group enriched genes and tissue enhanced genes in urinary bladder (Table 1). (C) Pie chart showing the distribution of the fraction of expressed mRNA molecules, i.e. the sum of all FPKM values for each of the categories for the genes expressed in urinary bladder, using the same color codes. (D) Network plot of the urinary bladder enriched gene (red) and group enriched genes (orange). Orange circle nodes represent a shared group of expressed genes and are connected to the respective enriched tissues (grey circles). The size of each orange node is related to the square root of the number of genes enriched in a particular combination of tissues.

Fig 2. Immunohistochemistry-based protein profiling of elevated genes in urinary bladder that are expressed in urothelium.

Of the 90 elevated genes in urinary bladder, 20 proteins are localized to the whole urothelium: CLEC3A (tissue enhanced gene), DHRS2 (group enriched gene), HOXA1 (tissue enhanced gene), PADI3 (group enriched gene), DUOXA2 (tissue enhanced gene), BMP3 (tissue enhanced gene), TRPA1 (tissue enhanced gene), HPGD (tissue enhanced gene), RBFOX3 (group enriched gene), ACER2 (tissue enhanced gene), CCR8 (tissue enhanced gene), CYP1A1 (group enriched gene), PTGS2 (tissue enhanced gene), OSTN (tissue enhanced gene), UPK1B (tissue enhanced gene), IL24 (group enriched gene), MYO3B (tissue enhanced gene), SHH (tissue enhanced gene), CTHRC1 (tissue enhanced gene), CHRDL2 (group enriched gene). All images are from The Human Protein Atlas and the title of each image shows the respective gene and antibody names.

Fig 3. Immunohistochemistry-based protein profiling of elevated genes in urinary bladder that are expressed in umbrella cells.

Of the 90 elevated genes in urinary bladder, UPK1A (group enriched gene), UPK2 (tissue enriched gene), UPK3A (group enriched gene) and UPK3B (group enriched gene) are localized specifically to umbrella cells in urothelium. All images are from the Human Protein Atlas and the title of each images show the respective gene and antibody names.

Fig 4. Immunohistochemistry-based protein profiling of elevated genes in urinary bladder that are expressed in intermediate/basal layer cells.

Of the 90 elevated genes in urinary bladder, KRT17 (tissue enhanced gene), PCP4L1 (tissue enhanced gene) and ATP1A4 (tissue enhanced gene) are localize specificity in intermediate/basal layer cells in urothelium. All images are from the Human Protein Atlas. Titles on each images are gene and antibody names of concerned proteins.

Fig 5. Examples of reverse analysis of proteins previously characterised as localized in urothelium.

Four proteins selected based on literature (GATA3, KRT5, MUC1, TP63) are stained in urothelium and agreed with literature: GATA3 and MUC1 in urothelium and KRT5 and TP63 in intermediate/basal cells. All images are from the Human Protein Atlas and the title of each image show the respective gene and antibody names.