Claudin-1 is a novel target of miR-375 in non-small-cell lung cancer

• We have identified CLDN1 as a novel target of miR-375 in non-small cell lung cancer.
• Overexpression of miR-375 down-regulates and knockdown of miR-375 up-regulates CLDN1 mRNA and protein, respectively.
• Overexpression of miR-375 promotes cell migration.
• High expression of miR-375 is associated with poor survival in non-small cell lung cancer.

ObjectivesWe previously reported low expression of miR-375 in squamous-cell carcinoma (SCC) and high expression in adenocarcinoma (AC) of the lung. miR-375's target genes and its function in non-small-cell lung cancer (NSCLC) have not been elucidated. Therefore, the present study was designed to identify the targets of miR-375 and to characterize its function in NSCLC.Materials and methodsCandidate targets of miR-375 were determined using a prediction database and previous data on differential gene expression between SCC and AC. We evaluated miR-375 and target-gene expression levels in 12 NSCLC cell lines. The effect of miR-375 overexpression and knockdown was evaluated in NSCLC cell lines by transfecting them with an miR-375 precursor or inhibitor. A luciferase-reporter assay was performed to confirm a direct interaction between miR-375 and its target gene. Further, a wound-healing assay was performed to evaluate the effect of miR-375 overexpression on the migration of SK-MES-1 cells. Finally, to assess the clinical relevance, 63 clinical NSCLC samples were analyzed.ResultsClaudin-1 (CLDN1) has 4 putative miR-375 target sites in its 3′-untranslated region, and this gene was determined to be a target of miR-375. CLDN1 messenger RNA and protein expression were attenuated by overexpression of miR-375 and increased by knockdown of miR-375 in NSCLC cell lines. In a luciferase-reporter assay, miR-375 overexpression resulted in a 3-fold repression of luciferase activity (P < 0.001). Cell migration was promoted by miR-375 overexpression, suggesting a high potential for invasion and metastasis in NSCLC expressing high levels of miR-375. In clinical NSCLC samples, there was a negative correlation between miR-375 and CLDN1 expression (r = −0.35, P = 0.005). In addition, high miR-375 expression was correlated with a shorter survival time among the clinical samples (P = 0.043).ConclusionCLDN1 is a novel target of miR-375, and high miR-375 expression shortens survival in NSCLC.