Abstract
The expression of neutrophil gelatinase-associated lipocalin (NGAL) is up-regulated in some cancers; therefore NGAL has potential as a tumor biomarker. Although the regulation mechanism for this is unknown, one study has shown that it is likely to involve a microRNA (miRNA). Here, we investigate the relation between miRNA expression and NGAL expression, and the role of NGAL in tumorigenesis. Using miRNA target–detecting software, we analyze the mRNA sequence of NGAL and identify a target site for microRNA-138 (miR-138) in nucleotides 25–53 of the 3′ UTR. We then analyze NGAL and miR-138 expression in three cancer cell lines originating from breast, endometrial and pancreatic carcinomas (the MCF-7, RL95-2 and AsPC-1 cell lines), respectively, using quantitative (real-time) PCR and western blot analysis. Metastasis is a critical event in cancer progression, in which malignant cell proliferation, migration and invasion increase. To determine whether miR-138-regulated NGAL expression is associated with metastasis, the proliferation and migration of the cell line are examined after miR-138 transfection. Using nude mice, we examine both the tumorigenicity of these cell lines and of miR-138-transfected cancer cells in vivo, as well as the effect of treating tumors with an antibody against NGAL. Our results show that these cancer cell lines down-regulate NGAL when miR-138 is highly expressed. Ectopic transfection of miR-138 suppresses NGAL expression and cell migration in RL95-2 and AsPC-1 cells, demonstrating that miR-138-regulated NGAL expression is associated with cell migration. Additionally, injection of the NGAL antibody diminishes NGAL-mediated tumorigenesis in nude mice, and miR-138 transfection of cancer cells reduces tumor formation. As the cell proliferation data showed that the tumor size should be regulated by NGAL-related cell growth. Taken together, our results indicate that NGAL may be a good target for cancer therapy and suggest that miR-138 acts as a tumor suppressor and may prevent metastasis.
Highlights
Neutrophil gelatinase-associated lipocalin (NGAL) was first isolated as a novel 25 kDa protein complex associated with human neutrophil gelatinase [1]
Complementarity Searching We evaluated microRNA target sequences in the 39 untranslated regions (39 UTRs) of the NGAL transcript using four different databases
A comparison of the results showed that the microRNA target sequence (CACCAGC) of miR-138 was present in the 39 UTR of NGAL (Fig. 1) in a higher estimated score than other microRNA, suggesting that transcriptional regulation of NGAL may be under miR-138 control
Summary
Neutrophil gelatinase-associated lipocalin (NGAL) was first isolated as a novel 25 kDa protein complex associated with human neutrophil gelatinase [1]. Leng et al [7] observed that the reduction of NGAL expression in breast cancer cells by injecting an antibody for NGAL inhibited tissue invasion by breast cancer cells and their migration. Down-regulation of NGAL expression in certain cancer cells or use of antibody neutralization against NGAL may, be effective in reducing metastasis and possibly provide a potential therapeutic approach for cancer. MicroRNAs (miRNAs) regulate various biological functions via post-transcriptional gene silencing. Their importance in the regulation of gene expression and, protein expression is correlated with disease formation in many organisms, as abnormal miRNA levels trigger gene up- or down- regulation in many cancers [8]. Because miRNA-suppressed genes may be involved in complex diseases or cancers, these small molecules could potentially be developed as diagnostic markers and therapeutic targets
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