Downregulation of CLDN7 due to promoter hypermethylation is associated with human clear cell renal cell carcinoma progression and poor prognosis

Abstract

BackgroundMetastasis is the primary cause of death in renal cell carcinoma (RCC). Loss of cell-to-cell adhesion, including tight junctions (TJs) is the initial step in the process of metastasis. Claudin-7 (CLDN7) is a major component of TJs. However, the clinical significance and its regulation of kidney tumorigenesis remain poorly understood.MethodsA total of 120 fresh clear cell RCC (ccRCC) specimens and 144 primary RCC and adjacent nonmalignant renal paraffin specimens were obtained from Department of Urology, Peking University First Hospital. Expression of CLDN7 in ccRCC tissues and cell lines were determined using bioinformatic data mining, quantitative real-time PCR (qRT-PCR), Western blotting and immunostaining. The clinical significance of CLDN7 expression and promoter DNA methylation status was analyzed in ccRCC patients from Peking University First Hospital and The Cancer Genome Atlas. Additionally, the methylation specific-PCR, bisulfite genomic sequencing and demethylation analysis of CLDN7 were performed. Biological functions of CLDN7 were investigated by examining cell proliferation using MTS assays and EdU incorporation assays, cell migration by in vitro wound healing assays and transwell migration assays, cell invasion by transwell invasion assays, and cell apoptosis by flow cytometry. Mouse model experiments were performed to confirm the effects of CLDN7 on tumor growth and metastasis in vivo. The molecular mechanism of CLDN7 function was investigated using gene-set enrichment analysis (GSEA) and high-throughput cDNA sequencing (RNA-Seq) and confirmed by qRT-PCR, Western blot and immunostaining in vitro and in vivo.ResultsOur findings revealed that CLDN7 is frequently downregulated via hypermethylation of its promoter in ccRCC. CLDN7 can help predict aggressive tumor status and poor prognosis in ccRCC patients. Interestingly, hypermethylation of the CLDN7 promoter was related to advanced ccRCC status and poor prognosis. Moreover, overexpression of CLDN7 induced cell apoptosis, suppressed proliferation, migration and invasion abilities of ccRCC cells both in vitro and in vivo. Additionally, GSEA and RNA-Seq results showed that CLDN7 had negative effects in cancer-associated signaling pathways and (epithelial-mesenchymal transition) EMT-related pathways. These results were validated by qRT-PCR, Western blot and immunostaining.ConclusionsWe have demonstrated a previously undescribed role of CLDN7 as a ccRCC suppressor and suggest that loss of CLDN7 potentiates EMT and tumor progression. CLDN7 may serve as a functional tumor suppressor in tumor progression and a potential biomarker and target in patients with ccRCC.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0924-y) contains supplementary material, which is available to authorized users.

Discovery and validation of the prognostic value of the lncRNAs encoding snoRNAs in patients with clear cell renal cell carcinoma

“…For example, hypermethylation of the CpG shore of the Shh gene resulted in Shh loss, and inhibition of DNA methylation increased Shh expression to halt the initiation of bladder cancer at the early stage of progression [48]; DNA methylation at an enhancer of the three prime repair exonuclease 2 gene (TREX2) was linked to decreased TREX2 gene expression and protein expression, which may affect drug-induced DNA damage repair in laryngeal cancer [49]; and Epigenetic Silencing of miRNA-338-5p and miRNA-421 drived SPINK1-Positive Prostate Cancer [50]. Besides, a recent study reported that downregulation of CLDN7 due to promoter hypermethylation contributed to human ccRCC progression and poor prognosis [51], indicating DNA methylation may also play vital roles in ccRCC. However, all of above studies have focused on the effect of DNA methylation on mRNA or miRNA, and the regulation of DNA methylation on lncRNA was rarely reported.…”

DOCK1 Regulates Growth and Motility through the RRP1B-Claudin-1 Pathway in Claudin-Low Breast Cancer Cells

“…A high level of DNMT expression leads to DNA hypermethylation and contributes to oncogenic activation [31]. Dysregulation of claudin expression has been shown to be associated with DNA hypermethylation status of the promoter region of certain genes [29,30,32,33,34]. Our data showed that DOCK1 depletion decreased DNMT1, DNMT3a, and DNMT3b, as well as global DNA methylation followed by claudin-1 up-regulation in CLBC.…”

“…Other members of the claudin family have also been reported to function as tumor suppressors. Overexpression of claudin-4 significantly reduces the invasive potential and clonogenic activity, as well as pulmonary metastasis, in human pancreatic cancer cells [28], breast cancer cells [29], and clear cell renal cell carcinoma [30]. Genes other than CLDN1 , CLDN3 , CLDN4 , CLDN7 , and ZO1 may also be coordinately regulated by DOCK1 and may contribute to tumor growth, but this remains to be investigated.…”

Low Expression of Claudin-7 as Potential Predictor of Distant Metastases in High-Grade Serous Ovarian Carcinoma Patients

“…Collectively, our data are consistent with a possible involvement of claudin-7 in ovarian cancer progression and, in the literature, the most recent publications support the pro-metastatic role of a diminished expression of claudin-7 in different type of epithelial cancers. For instance in colorectal cancer, where claudin-7 downregulation promotes the invasion and metastasis by regulating the EMT process (35), and in renal cancer, where downregulation of claudin-7 via hypermethylation of its promoter has been demonstrated and associated with metastatic features in vitro and in vivo (36).…”