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

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

Abstract

Background

Metastasis 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.

Methods

A 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.

Results

Our 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.

Conclusions

We 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.

Authors: Yifan Li†, Yanqing Gong†, Xianghui Ning, Ding Peng, Libo Liu, Shiming He, Kan Gong, Cuijian ZhangEmail author, Xuesong LiEmail author and Liqun Zhou

Influence Factor: 6.22

Citation: J Exp Clin Cancer Res 37, 276 (2018).

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