Overexpressed KCNK1 regulates potassium channels affecting molecular mechanisms and biological pathways in bladder cancer

Abstract

Background: This study aimed to explore the expression, molecular mechanism and its biological function of potassium two pore domain channel subfamily K member 1 (KCNK1) in bladder cancer (BC).

Methods: We integrated large numbers of external samples (n = 1486) to assess KCNK1 mRNA expression levels and collected in-house samples (n = 245) for immunohistochemistry (IHC) experiments to validate at the KCNK1 protein level. Single-cell RNA sequencing (scRNA-seq) analysis was performed to further assess KCNK1 expression and cellular communication. The transcriptional regulatory mechanisms of KCNK1 expression were explored by ChIP-seq, ATAC-seq and ChIA-PET data. Highly expressed co-expressed genes (HECEGs) of KCNK1 were used to explore potential signalling pathways. Furthermore, the immunoassay, clinical significance and molecular docking of KCNK1 were calculated.

Results: KCNK1 mRNA was significantly overexpressed in BC (SMD = 0.58, 95% CI [0.05; 1.11]), validated at the protein level (p < 0.0001). Upregulated KCNK1 mRNA exhibited highly distinguishing ability between BC and control samples (AUC = 0.82 [0.78-0.85]). Further, scRNA-seq analysis revealed that KCNK1 expression was predominantly clustered in BC epithelial cells and tended to increase with cellular differentiation. BC epithelial cells were involved in cellular communication mainly through the MK signalling pathway. Secondly, the KCNK1 transcription start site (TSS) showed promoter-enhancer interactions in three-dimensional space, while being transcriptionally regulated by GRHL2 and FOXA1. Most of the KCNK1 HECEGs were enriched in cell cycle-related signalling pathways. KCNK1 was mainly involved in cellular metabolism-related pathways and regulated cell membrane potassium channel activity. KCNK1 expression was associated with the level of infiltration of various immune cells. Immunotherapy and chemotherapy (docetaxel, paclitaxel and vinblastine) were more effective in BC patients in the high KCNK1 expression group. KCNK1 expression correlated with age, pathology grade and pathologic_M in BC patients.

Conclusions: KCNK1 was significantly overexpressed in BC. A complex and sophisticated three-dimensional spatial transcriptional regulatory network existed in the KCNK1 TSS and promoted the upregulated of KCNK1 expression. The high expression of KCNK1 might be involved in the cell cycle, cellular metabolism, and tumour microenvironment through the regulation of potassium channels, and ultimately contributed to the deterioration of BC.

Keywords: Biological functions; Gene expression; KCNK1; Molecular mechanism; Potassium channel.

Fig. 1

The research overflow of this study

Fig. 2

Comparison of differences in KCNK1 mRNA expression levels in control and bladder cancer groups. A KCNK1 mRNA was differentially expressed in each dataset. B Forest plot of standardised mean difference of KCNK1 mRNA in bladder cancer tissues. C Egger’s test. D Summary receiver operator characteristic

Fig. 3

KCNK1 protein levels were assessed by immunohistochemistry (IHC) assay based on in-house bladder cancer samples. A–E IHC staining of control samples. F–J IHC staining of cancer samples. K Protein levels of KCNK1 in bladder cancer and control samples. L Receiver operator characteristic curves of in-house IHC. ^ns/NSp > 0.05, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001

Fig. 4

Comprehensive analysis of single-cell RNA sequencing exploring KCNK1 expression. A Included cells were annotated into 8 cell types. B UMAP plot of KCNK1 expression in cells. C Violin plots of KCNK1 expression in different cells. D The cellular differentiation trajectory in pseudo-temporal analysis. E Expression levels of KCNK1 at cell differentiation. F Mapping of AUC values of each cell onto UMAP plots. G Number of cell interactions. H Cell interaction weights/strengths

Fig. 5

Cell communication analysis based on the ‘Cellchat’ package. A Ligand-receptor pair interactions in cellular communication. B Hierarchical structure of cell clusters involved in the MK signalling pathway. C Distribution of signalling genes involved in the MK signalling pathway. D Afferent signalling contributions of different cell clusters. E Different cell cluster efferent signalling contributions. F Signalling roles of different cell clusters in the MK signalling pathway

Fig. 6

Epigenetic regulatory mechanisms of KCNK1 expression. A Cistrome Data Browser-based transcription factor screening process. B Multi-omics exploration of potential regulatory mechanisms of KCNK1 (before amplification). C Multi-omics exploration of potential regulatory mechanisms of KCNK1 (after zoom-in). D Motif map of transcription factor GRHL2. E Motif map of the transcription factor FOXA1

Fig. 7

Pathway enrichment analysis of highly expressed co-expressed genes of KCNK1. A KEGG enrichment analysis. B Reactome enrichment analysis

Fig. 8

Preliminary validation of the molecular function of KCNK1. A Gene set enrichment analysis of KCNK1 in bladder cancer. B–I UMAP plots of single-cell metabolic activity scores

Fig. 9

Significance of KCNK1 expression in the tumour microenvironment (TME). A TME composition of the high KCNK1 expression group and the low KCNK1 expression group. B KCNK1 expression was significantly correlated with the level of multiple immune cell infiltration. C Correlation of KCNK1 expression with multiple immune cells. D Correlation of KCNK1 expression with TME scores. E Significant differences in TME scores between the high KCNK1 expression group and the low KCNK1 expression group. ^ns/NSp > 0.05, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001

Fig. 10

Clinical treatment and molecular docking of KCNK1 expression. A Immune-targeted therapy scores in the high KCNK1 expression group and low KCNK1 expression group. B Tumour immune escape scores in the high KCNK1 expression group and low KCNK1 expression group. C Assessment of the pharmacological therapeutic potential of KCNK1 expression. D Molecular docking to assess the affinity of KCNK1 protein to drugs