Down-regulation of NAMPT expression by miR-154 reduces the viability of breast cancer cells and increases their susceptibility to doxorubicin

Background Nicotinamide phosphoribosyltransferase (NAMPT) acts as an important enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. Deregulation of NAD could be associated with progression of many cancers including breast cancer. Here, we evaluated the effect of NAMPT inhibition via miR-154 on survival of breast cancer cells. Methods Breast cancer cell lines including MCF-7 and MDA-MB-231 were transfected with miR-154-5p mimic, inhibitor and their negative controls. Using real-time PCR and western blotting techniques the expression levels of NAMPT and miR-154 were determined and compared with the untreated cells. NAD levels were measured by an enzymatic method. Subsequently, colorimetric methods and ow cytometry were performed to evaluate cell viability and apoptosis. Bioinformatics analyses were performed to investigate whether NAMPT 3′-UTR is a direct target of miR-154 and the ndings were conrmed by luciferase reporter assay. Results According to the obtained results, NAMPT 3′-UTR was identied as a direct target of miR-154 and the levels of this miRNA was inversely associated with NAMPT expression both at mRNA and protein levels in breast cancer cell lines. Functionally, miR-154 inhibited the NAD salvage pathway leading to a remarkable decrease in cell survival and induction of apoptosis. Co-treatment of breast cancer cells with doxorubicin and miR-154 mimic signicantly reduced cell viability compared to treatment with doxorubicin alone in both cell lines. Conclusions Hence, it was concluded that the inhibition of NAD production by miR-154 might be introduced as a promising therapeutic strategy for the treatment of breast cancer either alone or in combination with other conventional chemotherapeutic agents. The cellular level of after transfection of MDA-MB-231 cells with different miR-154-related oligonucleotides compared to untreated cells. assay; diuoride microRNA response element; breast cancer; poly ADP-ribose polymerase 1; NC, negative control.

Among various cellular and molecular targets involved in breast cancer pathogenesis, microRNAs are proved to act as key epigenetic regulators (11). These molecules are known as a class of short noncoding RNAs that have effective roles in the adjustment of various biological functions including growth, angiogenesis, development, and differentiation (12). Studies have indicated that deregulation of miRNAs is directly related to the emergence of various aspects of tumorigenesis such as angiogenesis, tumor growth, metastasis, and response to therapy in breast cancer (13,14). miR-154 is a tumor suppressor which is located at chromosome 14q32 (14). Down-regulation of miR-154 is associated with progression of many cancers such as cancers of breast (14), prostate (15), osteosarcoma (16), hepatocellular carcinoma (17), thyroid (18) colorectal (19), and non-small cell lung cancer (20). However, the function and underlying cellular and molecular pathways related to miR-154 has not been determined in breast cancer. In this research, we accomplished bioinformatics analysis and found that in the 3′-UTR of NAMPT mRNA, there is a binding site for miR-154. Then, we investigated if the enhancement of miR-154 could reduce NAD levels and suppress breast cancer cell propagation via targeting NAMPT and whether this inhibition could modulate the cellular response to doxorubicin (DOX).

Methods
Cell lines and cell culture were also included in cell culture media. For culturing of MCF-10A cells, other supplements including insulin (10 μg/ml), hydrocortisone (0.5 μg/ml), epithelial growth factor (20 ng/ml) and cholera toxin (100 ng/ml) ( all from Sigma-Aldrich, Germany) were added up to MEGM. Finally, all cell lines were maintained at 37°C in an incubator that was humidi ed with water and contained 5% CO 2 .

Cell transfection
In order to perform cell transfection, polyethylenimine (PEI) (Sigma-Aldrich, Germany) was employed. To increase and decrease the level of miR-154-5p, we used microRNA mimic and microRNA inhibitor, respectively. These microRNAs were acquired from GenePharma (Shanghai, China). The sequences of miRNA mimic and inhibitor as well as their negative controls (i.e. NC inhibitor and NC mimic) are shown in table 1. Cells were seeded into 6-, 12-or 96-well plates and incubated as previously described. Then, fresh FBS-and antibiotic-free medium were added and the plates were incubated for another 4 hours. The PEI and oligonucleotides were mixed in Opti-MEM (Gibco, UK) and incubated for 40 min at 25°C and this mixture was subsequently added to each well followed by incubation of the plate at 37°C with 5% CO 2 for 4 h. In order to evaluate transfection, uorescein-conjugated miRNAs were used and the transfected cells were observed under a uorescence microscope (Olympus, Japan) 8-24 hours after transfection. ImageJ software (ImageJ, NIH, USA) was used for image analysis.

Cell survival assay
To evaluate the in uence of miR-154 mimic and its inhibitor on cell survival, viability assay was performed employing tetrazolium based WST-1 reagent (Roche Applied Science, Germany). A cell suspension containing total number of 5 × 10 3 cells/100 μl was seeded into every well of a 96-well plate.
After overnight incubation of cells, they were transfected using either mimic or inhibitor of miR-154. Relevant negative controls were also included in transfection experiments. Finally, WST-1 reagent (10 μl/well) was added and after 4 hours, optical density was measured by a plate reader (BioTek Instruments Inc., Winooski, USA) at 450/650 nm wavelength. For assessment of the effect of doxorubicin on cell viability, rst a 2 mg/ml stock solution of doxorubicin (Cell signaling technology, USA) in water was prepared. Subsequently, either un-transfected cells or cells transfected with different miR-154-related oligonucleotides were treated with 0.1 μM concentration of doxorubicin for 24 hours and the cell survival was evaluated as described above.

Apoptosis assay
The effect of miR-154 on cell apoptosis was assessed by employing a ow-cytometric detection kit containing FITC-Annexin V and propidium iodide (PI) (Roche Applied Science, Germany) following the instructions provided by the kit. A total of 3×10 5 of MCF-7 and MDA-MB-231 cells were seeded in 6-well plates and after 48 hours, cells were harvested and after washing twice with PBS, were stained with Annexin V as well as PI. Finally, evaluation of stained cells was done by ow-cytometry (FACScan, BD Biosciences, USA) equipped with bandpass lters at 515 nm for detection of FITC, 600 nm for PI detection and 488 nm laser for excitation.
CellQuest software (BD Biosciences) was employed for estimation of obtained results. The cells that were positive for Annexin V-FITC were presented as cells that had undergone apoptosis.

Western blotting
Western blot technique was used to investigate the effect of miR-154 alterations on NAMPT expression levels in the breast cancer cell lines. After 48 hours, RIPA lysis buffer containing protease inhibitor (0.1%), phosphatase inhibitor (0.5%) and phenylmethane sulfonyl uoride (PMSF) (10% ) (all from Sigma-Aldrich, Germany) was used for cell lysis. Then, lysates (40 µg of the total protein from each sample) was subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) on polyacrylamide gel (10%). In the next step, separated proteins were trans-blotted onto polyvinylidene di uoride (PVDF) membrane. After blocking of membranes in blocking buffer containing 0.05% Tween 20 and 5% powdered non-fat milk in PBS, the membranes were incubated with a PBEF/NAMPT Rabbit antibody at 1:1000 concentration (cell signaling technology, USA) followed by incubation with HRP-conjugated secondary antibody against rabbit IgG, at a dilution of 1:5000 (Cell Signaling Technology, USA). NAMPT protein and GAPDH (control) bands were detected by enhanced chemiluminescent reagent (Amersham Biosciences/GE Healthcare, U.K). Analysis of the resulting bands was performed by densitometry using ImageJ software (v1.52, NIH). For this purpose, the rst band was primarily selected by drawing a rectangular shape around it and assigning it as the rst lane. Then it was proceeded by selection of the other bands by moving the same rectangle to make sure that all selections have the same dimensions. Subsequently, the lane pro le plot was generated. The area under the curve of each plot was determined which corresponded to the density of each band.

NAD level assay
The concentration of intracellular NAD/NADH was measured using NAD assay kit (Abcam, UK) according to instructions provided by the manufacturer. After lysis of the transfected cells using lysis buffer, the concentration of the total protein in the lysate was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scienti c, USA). Subsequently, the proteins were removed using perchloric acid and the NAD levels were obtained by measuring the absorbance at 450 nm. The quantity of NAD was normalized against the protein content in each sample.

Investigation of miR-154-target interaction
To check the direct interaction of miR-154 with NAMPT 3'-UTR, the sequence of the target microRNA binding sites was cloned in the psiCHECK-2 reporter plasmid as previously described (21). Brie y, the NAMPT 3'-UTR sequence was rst determined using Gene database of PubMed (https://www.ncbi.nlm.nih.gov/gene). The relevant region was ampli ed by the primers listed in Supplementary Table 1; synthesized by Macrogen Inc., South Korea. The tandem mutant of NAMPT 3′-UTR was also constructed to serve as a negative control. To create this sequence, the forward and reverse primers containing restriction sites for NotI and XhoI were used (Supplementary Table 1). To ensure the presence of fused sequences and the absence of mutations in the sequence, the recombinant plasmid of NAMPT-MRE-tandem-mut-psiCHECK2 was sequenced by Macrogen Inc. The recombinant constructs were co-transfected with different miR-154-related oligonucleotides into HEK293T cell line. The activity of Renilla luciferase was measured over the activity of re y by dual luciferase assay kit obtained from Promega, (USA).

Statistical analysis
All statistical analyses were done using GraphPad Prism software, version 5.01 (USA, San Diego). The differences between the experimental groups were evaluated by one way-ANOVA. All results were presented as mean ± standard deviation (S.D.). P values lower than 0.05 were recognized statistically signi cant.

Results
The expression levels of miR-154 and NAMPT in breast cancer cell lines WST-1 cell survival assay. Survival rate of (a) MCF-7 and (b) MDA-MB-231 cells in the response to increased and decreased levels of miR-154 by its mimic and inhibitor, respectively. The obtained results are expressed as percentage to untreated control. Data are mentioned as mean ± SD of triplicate experiments that were repeated at least three times. * P<0.05, ** P <0.01.

miR-154 increased the susceptibility of breast cancer cells to doxorubicin
Considering the effect of miR-154 on cell viability, we treated the studied cell lines with doxorubicin after transfection. As it is shown in gure 7, when miR-154 mimic was used in combination with doxorubicin, the cell viability was signi cantly diminished compared to either doxorubicin or miR-154 mimic alone. This effect was observed in both MCF-7 and MDA-MB-231 cells (Figure 7 a, b). On the contrary, downregulation of cellular miR-154 by its inhibitor led to a lower response to doxorubicin treatment and the cell viability in this group (miR-154 inhibitor + doxorubicin) was similar to untreated control cells (Figure 7). miR-154 regulated NAMPT by direct binding to its 3′-UTR As previously stated, bioinformatics analysis showed that miR-154 is among the miRNAs that are conserved among mammals and it was predicted that 3′-UTR region of NAMPT mRNA could be a potential target for miR-154. To con rm this, the luciferase reporter activity of psiCHECK2 vector having NAMPT-related 3′-UTR in the presence of desired oligonucleotides was investigated. miR-154 mimic decreased the luciferase activity by 59.5 ± 0.03% compared to untreated control cells (P <0.01); however, miR-154 inhibitor led to a signi cant increase in luciferase activity (P <0.05) (Figure 9). None of the controls signi cantly affected the luciferase activity.
Luciferase reporter assay verifying the predicted interaction between miR-154 and 3′-UTR of NAMPT. PsiCHECK2 vector harboring NAMPT 3′-UTR or the mutant form of miR-154 recognition element (NAMPT-MRE-tandem-mut) were co-transfected with microRNA-related oligonucleotides into HEK293T. Fire y luciferase activity was normalized with respect to Renilla luciferase as control. The experiments were repeated at least three times. * P<0.05, ** P <0.01.

Discussion
Over 2 million new cases of breast cancer have been reported in 2018 (23). Therefore, novel methods including strategies based on cancer-related molecular changes are essential for breast cancer management. In this research, we indicated that the miR-154 expression was remarkably reduced in breast cancer cell lines in comparison with normal mammary cells. Consistently, Qin et al. studied the role of miR-154 in breast carcinogenesis and showed that miR-154 was down-regulated in this malignancy and increased cellular proliferation and metastasis potential through targeting ADAM metallopeptidase domain 9 (ADAM9) (24). In another study by Xu et al., E2F transcription factor 5 protein (E2F5) was introduced as a direct target of miR-154, with reciprocal relationship between these two parameters in in neoplastic cells of breast (25). Additionally, reduced levels of miR-154 and its association with aggressive clinicopathological characteristics have been reported in glioma, colorectal, prostate and non-small cell lung cancers (26-29).
In the current study, a negative correlation was found between miR-154 and NAMPT expression (underand over-expressed, respectively) in breast cancer cells, suggesting the inhibitory effect of miR-154 on NAMPT expression. Signi cantly higher expression of NAMPT in breast cancer tissues compared with normal mammary gland tissue has been previously reported and has been shown to be related to a higher tumor growth, advanced clinical stages, increased expression of progesterone and estrogen receptors and lymph node metastasis. Meanwhile, over expression of NAMPT in patients results in poor overall or disease-free survival (21,(30)(31)(32)(33)(34)(35). Additionally, exogenous administration of recombinant NAMPT not only leads to increased cell proliferation by activation of signaling pathways, but also increases cell survival by NAD production (36, 37).
The results achieved in our study explained that the up-regulation of miR-154 signi cantly suppressed NAMPT expression both at mRNA and protein levels; indicating that this interaction leads to mRNA degradation or suppression of translation as has been suggested as mechanisms of action of miRNAs (28). The regulatory effect of miR-154 was further con rmed by reducing its cellular levels using miR-154 inhibitor which in turn led to a signi cant rise in NAMPT expression levels. The obtained results from luciferase reporter assay demonstrated that the aforementioned regulatory outcome of miR-154 on NAMPT was directly the effect of binding of miR-154 to the NAMPT 3´-UTR, ruling out the possibility of off-targets and indirect effects. These ndings are consistent with our recent study in which, miR-206 was introduced as a potential inhibitor of NAD biosynthesis in HBC cells via direct binding to NAMPT 3´-UTR (21). Consistently, NAMPT 3´-UTR has been reported in a number of studies, as the potential target for a wide variety of miRNAs in other malignancies and diseases including miR-182 in the ossi cation of ligamentum avum (38), miR-300 in neonatal sepsis (39), miR-34a in obesity (40), miR-206 in pancreatic cancer (41), miR-26b in colorectal cancer (42), miR-410 in pulmonary arterial hypertension (PAH) (43) and miR-182 in HIV-1 contaminated cells (44).
The ndings of NAD measurement revealed that miR-154 caused the attenuation of intracellular NAD via inhibition of NAMPT salvage pathway. NAMPT is an essential enzyme in NAD biosynthesis and therefore its inhibition is a plausible approach in depleting intracellular NAD (30,45). Here we showed that inhibition of NAMPT and the further decline in NAD levels leads to diminished cell viability and a prominent induction of apoptosis. We had earlier reported that inhibition of NAMPT by its speci c inhibitor could effectively reduce NAD levels and induce apoptosis in breast cancer cells (46). In line with our ndings, inhibition of NAMPT by microRNAs has also been shown to be an appropriate method in modulating NAD content and cell viability (40)(41)(42).
One major obstacle in cancer therapy is chemoresistance to the commercial cancer therapeutic drugs, contributing to cancer progression, recurrence and mortality (47). Aberrant miRNA expression is related to anti-cancer drug resistance (47). In the current study, we revealed that miR-154 increased the cellular response to DOX and cell survival was signi cantly decreased when DOX treatment was combined with up-regulation of miR-154. Consistently, up-regulation of miR-760 has been shown to be able to sensitize breast cancer cells to the anti-cancer drugs (48). miR-133b has also been found to be functionally involved in the DOX resistant breast cancer cells and the intra-tumoral delivery of this miRNA increased the treatment response to DOX in DOX-resistant xenografts (49). NAMPT overexpression has been shown to be associated with low response to chemotherapeutic agents including doxorubicin, etoposide, uorouracil, paclitaxel, and phenylethyl isothiocyanate (50). Thus, over-expression of miR-154 might be an e cacious approach to increase the sensitivity of breast cancer cells to DOX treatment.

Conclusions
The obtained ndings of this research suggest that the role of miR-154 in the inhibition of NAMPTmediated NAD production pathway via targeting NAMPT 3′-UTR is crucial. Overexpression of this miRNA reduces the survival of breast cancer cells and stimulates apoptosis in these cells. As a result, this molecular mechanism can be employed as a promising method for breast cancer treatment. Availability of data and materials

Abbreviations
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.     WST-1 cell survival assay. Survival rate of (a) MCF-7 and (b) MDA-MB-231 cells in the response to increased and decreased levels of miR-154 by its mimic and inhibitor, respectively. The obtained results are expressed as percentage to untreated control. Data are presented as mean ± SD of triplicate experiments that were repeated at least three times. * P<0.05, ** P <0.01.