Significance and Possible Biological Mechanism for CLDN8 Downregulation in Kidney Renal Clear Cell Carcinoma Tissues

Abstract

Background: The clinical role of claudin 8 (CLDN8) in kidney renal clear cell carcinoma (KIRC) remains unclarified. Herein, the expression level and potential molecular mechanisms of CLDN8 underlying KIRC were determined.

Methods: High-throughput datasets of KIRC were collected from GEO, ArrayExpress, SRA, and TCGA databases to determine the mRNA expression level of the CLDN8. In-house tissue microarrays and immunochemistry were performed to examine CLDN8 protein expression. A summary receiver operating characteristic curve (SROC) and standardized mean difference (SMD) forest plot were generated using Stata v16.0. Single-cell analysis was conducted to further prove the expression level of CLDN8. A clustered regularly interspaced short palindromic repeats knockout screen analysis was executed to assess the growth impact of CLDN8. Functional enrichment analysis was conducted using the Metascape database. Additionally, single-sample gene set enrichment analysis was implied to explore immune cell infiltration in KIRC.

Results: A total of 17 mRNA datasets comprising 1,060 KIRC samples and 452 non-cancerous control samples were included in this study. Additionally, 105 KIRC and 16 non-KIRC tissues were analyzed using in-house immunohistochemistry. The combined SMD was -5.25 (95% confidence interval (CI): -6.13 to -4.37), and CLDN8 downregulation yielded an SROC area under the curve (AUC) close to 1.00 (95% CI: 0.99 - 1.00). CLDN8 downregulation was also confirmed at the single-cell level. Knocking out CLDN8 stimulated KIRC cell proliferation. Lower CLDN8 expression was correlated with worse overall survival of KIRC patients (hazard ratio of CLDN8 downregulation = 1.69, 95% CI: 1.2 - 2.4). Functional pathways associated with CLDN8 co-expressed genes were centered on carbon metabolism obstruction, with key hub genes ACADM, ACO2, NDUFS1, PDHB, SDHD, SUCLA2, SUCLG1, and SUCLG2.

Conclusions: CLDN8 is downregulated in KIRC and is considered a potential tumor suppressor. CLDN8 deficiency may promote the initiation and progression of KIRC, potentially in conjunction with metabolic dysfunction.

Keywords: CLDN8; Kidney renal clear cell carcinoma; Mechanism; Prognosis; Tissue microarray.

Figure 1

Flow chart of mRNA expression datasets for kidney renal clear cell carcinoma tissues.

Figure 2

Immunohistochemical staining of claudin 8 (CLDN8) protein in non-KIRC and KIRC tissues. (a, b) Non-KIRC tissues. (c, d) KIRC tissues. KIRC: kidney renal clear cell carcinoma.

Figure 3

(a-d) Immunohistochemistry images of claudin 8 (CLDN8) protein in kidney renal clear cell carcinoma tissues derived from the human protein atlas (HPA) database.

Figure 4

The decline in claudin 8 (CLDN8) expression in kidney renal clear cell carcinoma. (a) Forest plot depicting significant downregulation of CLDN8 expression in kidney renal clear cell carcinoma tissues. (b) Egger’s plot and (c) Begg’s funnel plot indicate the absence of publication bias (P = 0.09).

Figure 5

Integrated analysis of effect size forest plots, summary receiver operating characteristics curve, and survival analysis. (a) Sensitivity and specificity assessment. (b) Summary receiver operating characteristics curve. (c) Positive and negative likelihood ratios. (d) Kaplan-Meier survival curve.

Figure 6

Expression profiling of claudin 8 (CLDN8) in single cells of kidney renal clear cell carcinoma. (a) Cell distribution in kidney renal clear cell carcinoma (KIRC). (b) Comparative analysis of CLDN8 mRNA expression levels in KIRC cells and normal cells.

Figure 7

Gene effect score analysis of claudin 8 (CLDN8) in 20 kidney renal clear cell carcinoma cell lines. The findings suggest a potential inhibitory role of CLDN8 in the growth of kidney renal clear cell carcinoma cells.

Figure 8

Functional enrichment analysis of claudin 8 (CLDN8) and hub genes. (a) Gene ontology terms encompassing biological processes, cell components, and molecular functions. (b) Kyoto encyclopedia of genes and genomes pathways. (c) Identification of top 10 hub genes via protein-protein interaction network analysis using Cytohubba.

Figure 9

Survival analysis of hub genes and several immune cells. (a) Downregulation of eight hub genes, including ACADM, ACO2, NDUFS1, PDHB, SDHD, SUCLA2, SUCLG1, and SUCLG2, correlates with shorter overall survival of kidney renal clear cell carcinoma (KIRC) patients. (b) Functional interaction network of the eight hub genes using GeneMANIA algorithm. (c-g) Correlation between infiltration levels of various immune cells and survival of KIRC patients. (c) Activated CD4⁺ T cells. (d) Effector memory CD8⁺ T cells. (e) Regulatory T cells. (f) Macrophage. (g) CD56bright natural killer cells.