Renal cell carcinoma (RCC) is one of the most common tumors of the urinary system, accounting for 3–5% of adult malignant tumors in recent years . ccRCC accounts for up to 80% of all kidney cancers . Despite continuous in cancer treatment, the prognosis of patients with advanced and metastatic ccRCC remains poor, and the mortality rates continue to increase . Approximately 20–30% of patients with ccRCC have already experienced local or systemic metastasis when clinically diagnosed [15, 20]. Under the existing conditions, the diagnosis and treatment of ccRCC is still one of the more difficult problems. Therefore, it is particularly important to explore the genes related to the development of ccRCC and discover new molecular markers suitable for clinical application.
CLDNs are the main components of tight junctions in epithelial cells, and have functions, such as maintaining cell junctions, polarity, and signal transduction, thus regulating cell proliferation, apoptosis, migration and invasion [21, 22]. Moreover, the dysregulation of CLDN expression is closely related to the occurrence and development of many malignant tumors. CLDN2 inhibits ccRCC progression by inhibiting YAP-activation . In bladder cancer, loss of CLDN11 may be associated with proliferation, recurrence, and invasiveness . Reduced CLDN12 expression predicts poor prognosis in cervical cancer . CLDN15 is a novel diagnostic marker of malignant pleural mesothelioma . However, the expression levels of the CLDN family members in ccRCC and effect of their differential expression on ccRCC development remain unknown.
In this study, we found seven CLDN family members with low expression in ccRCC tissues compared with that in healthy kidney tissue samples, namely CLDN7, 8, 10, 11, 14, 16, and 19. They were significantly associated with the cancer stage and tumor grade in patients with ccRCC. With the development of tumors, its expression level decreases, indicating that it plays a role as a tumor suppressor gene. It has been reported that CLDN8 could serve as an independent prognostic factor in ccRCC, in which it suppresses 786–O proliferation, migration, and invasion through the EMT and AKT pathways . This finding is consistent with the results of our analysis. The Kaplan-Meier Plotter database was used to analyze the effects of CLDN7, 8, 10, 11, 14, 16, and 19 on the survival rate of patients with ccRCC. The results indicated that the OS rate of patients with ccRCC with low CLDN8, 10, 11, and 14 expression was significantly reduced. These results were verified by the UALCAN database, suggesting that patients with low CLDN7, 8, 10, 11, and 14 expression had significantly lower OS rates. CLDN16 and CLDN19 also showed similar characteristics in patients with ccRCC. Although their expression in tumor tissues is lower than that in normal tissues, it needs to be further verified whether CLDN16 and CLDN19 play an inhibitory role in ccRCC, similar to other CLDN family members.
As we all know, methylation is an important modification of protein and nucleic acid, regulating gene expression and shutdown, and is one of the important contents of epigenetics. Studies have shown that abnormal methylation leads to the instability in gene expression and transcription, which is an important reason for the occurrence and development of tumors . Epigenetic modifications, such as methylation of CLDNs have been reported to be promising prognostic markers in various cancers [29, 30]. Therefore, the methylation levels of CLDN7, 8, 10, 11, 14, 16, and 19 were analyzed using the UALCAN database, and the results showed that their methylation levels were significantly increased in ccRCC. Furthermore, studies have shown that the downregulation of CLDN7 induced by promoter hypermethylation is associated with progression and poor prognosis in human ccRCC . Related studies have shown that DNA hypermethylation is an important factor in the decreased expression of CLDN10 in ccRCC . These results are consistent with our findings.
Cancer is currently regarded as a disease characterized by uncontrolled cellular growth caused primarily by genetic alterations that mainly occur in a set of cancer driver genes, conferring transformed cells with certain selective advantages over neighboring cells . For example, mutations in CLDN1, 5, 14, 16, and 19 have been reported to cause neonatal ichthyosis, sclerosing cholangitis, non-syndromic deafness, familial hypomagnesemia, and other symptoms in humans . We investigated the genetic variation of the CLDN family in ccRCC using the cBioPortal for Cancer Genomics. Twenty-four genes were queried in 512 ccRCC samples (TCGA, Pan-Cancer Atlas), and these queried genes were altered in 244 (48%) samples. However, whether these genetic alterations are the drivers of ccRCC, whether they contribute to tumorigenesis, and the exact tumorigenesis mechanism remain to be determined. CLDNs expression in cancer is regulated by factors other than genetic alterations.
Next, using String-DB database mining analysis, CLDN7, 8, 10, 11, 14, 16, and 19 interacted with CLDN24, CLDN15, CDY1, CLDN20, CLDN22, and CLDN23. They are mainly involved in biological processes, such as tight junction-specific obliteration of the intercellular space, cellular barriers, and tight connections between cells. GeneMANIA results also revealed that the functions of the differentially expressed CLDNs and their associated molecules (such as CLDN24, CLDN15, CDY1, CLDN20, CLDN22, CLDN23) were primarily related to tight junction organization, tight junction, and apical junction assembly. Pathway enrichment analyses of interacting proteins using the Metascape database showed that CLDN7, 8, 10, 11, 14, 16, and 19 were mainly involved in epithelial cell differentiation, cell morphogenesis, and cell-cell junction organization. Disruption of CLND strands allows foreign molecules and microorganisms to enter tissues through the paracellular space, causing inflammation, which is the most common predisposing factor for cancer . Moreover, the disruption of CLDN strands also allows growth factor infiltration into the mucosa to promote neoplastic transformation and growth . These results indicated that the downregulated expression of CLDN7, 8, 10, 11, 14, 16, and 19 in ccRCC directly damages and affects the function of tightly coupled structures.
TME mainly includes stromal and immune cells, and the degree of infiltration of immune cells is highly correlated with the occurrence, development, and immunotherapy response of tumors . In this study, TIMER analysis revealed that the expression of CLDNs may be significantly correlated with the infiltration of six immune cell types, indicating that CLDNs are closely related to tumor immune cell infiltration and are also involved in the pathogenesis of ccRCC. Our study provides a theoretical basis for the design of novel immunotherapies.
In summary, we systematically analyzed the expression and prognostic value of CLDNs in ccRCC. Our findings suggest that CLDN8, 10, 11, and 14 could be used as a biological markers for prognosis evaluation and malignancy analysis of ccRCC. However, our experiment was only at the data analysis level and should be verified experimentally.