Up-regulation of miR-381 inhibits NAD+ salvage pathway and promotes apoptosis in breast cancer cells
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Date
2019-08-27
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Abstract
Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme involved in nicotinamide adenine di-
nucleotide (NAD) salvage pathway, is overexpressed in many human malignancies such as breast cancer. This
enzyme plays a critical role in survival and growth
of cancer cells. MicroRNAs (miRNAs) are among the most
important regulators of gene expression, and serve as potential targets for diagnosis, prognosis, and therapy of
breast cancer. Therefore, the aim of this study was to asse
ss the effect of NAMPT inhibition by miR-381 on breast
cancer cell survival. MCF-7 and MDA-MB-2
31 cancer cell lines were transfected
with miR-381 mimic, inhibitor,
and their corresponding negative controls (NCs). Subsequently, the level of NAMPT and NAD was assessed using real-time PCR, immuno-blotting, and enzymatic methods, resp
ectively. In order to evalua
te apoptosis, cells were
labelled with Annexin V-FITC and propidium iodide and analyzed by flow cytometry. Bioinformatics analysis
was performed to recognize whether NAMPT 3
′
-untranslated region (UTR) is a direct target of miR-381 and the
results were authenticated by the luciferase re
porter assay using a vector containing the 3
′
-UTR sequence of
NAMPT. Our results revealed that the 3
′
-UTR of NAMPT was a direct target of miR-381 and its up-regulation
decreased NAMPT gene and protein expression, leading to a notable reduction in intracellular NAD and subse-
quently cell survival and induction of apoptosis. It can be concluded that miR-381 has a vital role in tumor sup-
pression by down-regulation of NAMPT, and it can be a promising candidate for breast cancer therapy.
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Keywords
Nicotinamide phosphoribosyltransferase, Nicotinamide adenine dinucleotide, miR-381, Apoptosis, Breast cancer, Tumor suppression