Abstract
BackgroundIn an earlier study, we investigated the expression of tight junction protein claudins (CLDNs) in human osteosarcoma (OS) cells, and the CLDN2 was found to be down-regulated in primary tumor cells compared with normal osteoblast cells. Here, we sought to explore the effects of CLDN2 on the malignant phenotype of OS and the underlying molecular mechanisms.MethodsThe expression patterns of CLDN2 and afadin in OS tissues and histologically non-neoplastic bone tissues were explored via immunohistochemistry and western blotting. CLDN2 expression levels in an OS cell line stably expressing CLDN2 and an osteoblast cell line with a CLDN2 knockout were confirmed by western blotting and immunofluorescence staining. The malignant phenotype of OS cells and osteoblast cells in vitro was assessed using a cell counting kit-8 assay, transwell assay and wound-healing experiment. Western blotting was utilized to detect the activation state of Ras/Raf/MEK/ERK pathway. Moreover, an RNA interference method were used to silence afadin in CLDN2-expressing OS cells.ResultsOur research group found that CLDN2 and afadin was underexpressed in OS tissues, and the overexpression of CLDN2 significantly inhibited the migration abilities of OS cells. Genetic silencing of afadin in CLDN2-overexpressing OS cells promoted U2OS cell motility and activation of the Ras/Raf/MEK/ERK pathway.ConclusionsIn this study, we confirmed that CLDN2 expression significantly inhibited the malignant phenotype of OS cells in vitro. Inhibition of the ERK pathway by afadin may be one of the mechanisms by which CLDN2 blocks the metastasis phenotype of OS cells.
Highlights
In an earlier study, we investigated the expression of tight junction protein claudins (CLDNs) in human osteosarcoma (OS) cells, and the CLDN2 was found to be down-regulated in primary tumor cells compared with normal osteoblast cells
It has been reported that CLDN1 is a metastasis suppressor for lung adenocarcinoma and that a lack of CLDN1 expression is associated with poor patient prognosis [12]
Expressions of CLDNs family members in OS cell lines and osteoblasts Real-time quantitative PCR and western blotting were used to detect the expression of CLDNs family members in fetal-osteoblast cell line, hFOB.1.19 and OS cells U2OS, Saos2
Summary
We investigated the expression of tight junction protein claudins (CLDNs) in human osteosarcoma (OS) cells, and the CLDN2 was found to be down-regulated in primary tumor cells compared with normal osteoblast cells. Previous studies have shown that for various human malignancies, metastasis is accompanied by abnormalities in tight junction (TJ). Studies by Usami et al indicated that reduced CLDN7 expression in head and neck cancer cells promoted cell invasion and migration [13]. These studies indicate that specific expression patterns of CLDNs in tumors have the potential to be used as molecular markers of malignancies
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