The expression levels of miR-154 and NAMPT in breast cancer cell lines
Figure 1a shows the relative expression of miR-154 in untreated MDA-MB-231 and MCF-7 cell lines compared to normal epithelial cell line (MCF-10A) that was used as control. It can be observed that miR-154 expression levels were considerably lower in MDA-MB-231 and MCF-7 (both P<0.01) cell lines in comparison with MCF-10A cells. Additionally, measurement of the mRNA expression of NAMPT indicated that NAMPT was expressed higher in MDA-MB-231 (P<0.05) and MCF-7 (P<0.01) cell lines as opposed to MCF-10A (Figure 1b). Furthermore, the blotting results showed that the basal level of NAMPT protein in MDA-MB-231 and MCF-7 and cell lines was higher than MCF-10A (P values less than 0.05 and 0.01, respectively) (Figure 1 c, d).
The expression level of miR-154 and NAMPT in un-transfected cells. Basal expression levels of (a) miR-154 and (b) NAMPT were compared with those in MCF-10A cells. Each vertical bar represents the mean ± SD of triplicate determinations. *P<0.05; **P<0.01. (c) Evaluation of NAMPT basal expression at protein level by immunoblotting and (d) the quantification of the resulting bands by densitometry. Each bar is the mean ± SD of at least three independent experiments. *P<0.05; **P<0.01.
miR-154 cellular levels was up-regulated via miRNA mimic transfection
In order to clarify the mechanism by which miR-154 controls NAMPT expression, transfection experiments were conducted. Cellular transfection was conducted with either miR-154 mimic which was expected to increase the intracellular levels of miR-154 or its antisense oligonucleotide serving as miR-154 inhibitor to sequester or decrease miR-154. The MCF-7 cell line transfected with the mimic showed a significant increase in miR-154 levels (P<0.01), while, a decline in miR-154 expression was observed following transfection with its inhibitor (P<0.001) (Figure 2a). The MDA-MB-231 cells also exhibited a significantly enhanced cellular levels of miR-154 after transfection with miRNA-mimic (P<0.001). In contrast, transfection of MDA-MB-231 cells with miR-154 inhibitor was associated with a remarkable decrease in miR-154 level (P<0.001) (Figure 2b). Fluorescence microscopy results of the cells transfected with fluorescein-labeled microRNAs confirmed successful transfection (Supplementary figure 1).
Relative expression of miR-154 after transfection of breast cancer cells. The cellular level of miR-154 after transfection of (a) MCF-7 and (b) MDA-MB-231 cells with different miR-154-related oligonucleotides compared to untreated control cells. **P <0.01, *** P <0.001.
miR-154 and NAMPT gene expression
As described earlier, bioinformatics analysis anticipated that 3′-UTR of NAMPT is potentially targeted by miR-154. So, it was supposed that down-regulated miR-154 in cancer cells might be involved in NAMPT up-regulation. To evaluate whether miR-154 would exert an inhibitory effect on NAMPT expression, RT-PCR were performed on transfected human breast cancer cells. At the mRNA level, NAMPT gene revealed a significantly reduced expression in both breast cancer cell lines (P<0.001) due to miR-154 augmentation by its mimic. Quite the reverse, blocking miR-154 by its corresponding inhibitor caused a significant increase in the expression of NAMPT mRNA in both of the studied cell lines (P<0.001 and P<0.05, respectively) (Figure 3 a, b).
NAMPT gene expression in breast cancer cells after transfection. Relative NAMPT mRNA expression in (a) MCF-7 and (b) MDA-MB-231 cells transfected with miR-154 mimic, miR-154 inhibitor or their negative controls (NC) compared to untreated cells. Each column represents the mean ± SD of at least three separate experiments. *P<0.05; ***P<0.001
Suppression of NAMPT protein expression by miR-154
The results obtained from Western blotting experiments indicated that the up-regulation of miR-154 via transfection with miR-154 mimic, remarkably reduced the levels of NAMPT protein in MCF-7 (P<0.05) as well as MDA-MB-231 (P<0.05) cells (Figure 4a, 4b). Nevertheless, NAMPT protein expression was enhanced in both MCF-7 (P<0.01) and MDA-MB-231 (P<0.001) cell lines following transfection with miR-154 inhibitor (Figure 4a, 4b).
Suppression of NAMPT protein expression by miR-154.
Quantitation of NAMPT protein level in (a) MCF-7 and (b) MDA-MB-231 cells transfected with the mimic of miR-154 or its inhibitor. Negative controls (NC) were also used for transfection. The results were compared to untreated control. Graphs represent the mean± SD of the results of the densitometric analysis of the blotting images normalized to GAPDH as the internal control and presented relative to those in control cells. Representative immunoblot images of NAMPT protein measurement in (a) MCF-7 and (b) MDA-MB-231 cells. *P<0.05, **P<0.01, *** P <0.001.
The effect of miR-154 on NAD depletion
Increased NAMPT level is correlated with high concentration of NAD in malignant cells (5). Our results showed that NAD was decreased in the MCF-7 cells that were transfected with the mimic of miR-154 compared to un-transfected control cells (P<0.001). On the contrary, there was a significant augmentation of NAD level in the cells that were transfected with miR-154 inhibitor (P<0.05) (Figure 5a). Similarly, the NAD level in MDA-MB-231 cells transfected with the mimic of miR-154 exhibited a significant increase (P<0.01), while a considerable decrease in NAD was observed in those transfected with miR-154 inhibitor (P<0.05) (Figure 5b).
The effect of miR-154 on intracellular NAD levels. Evaluation of relative NAD levels in (a) MCF-7 and (b) MDA-MB-231 cell lines after transfection with miR-154 mimic, inhibitor or their negative controls (NC) compared to un-transfected control. Results are presented as mean ± SD from three duplicate experiments that were performed separately. *P<0.05, ** P<0.01, ***P<0.001.
Increase of miR-154 in breast cancer cells reduced cell viability
NAMPT is elevated in diverse human malignancies such as breast cancer. This enzyme facilitates proliferation and increases survival of cancer cells (22). In the present research, we studied the effect of miR-154 on the survival of breast cancer cells using WST-1 cell survival assay. The obtained results revealed that miR-154 mimic considerably reduced cell survival in MCF-7 (P<0.05) and MDA-MB-231 (P<0.01) cells when compared to the untreated cells; whereas, treating the cells with miR-154 inhibitor considerably enhanced cell survival in both cell lines (both P<0.01). The obtained results are shown in figure 6.
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 figure 7, when miR-154 mimic was used in combination with doxorubicin, the cell viability was significantly 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, down-regulation 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).
Effects of miR-154 on susceptibility of breast cancer cells to doxorubicin. The effect of miR-154 and doxorubicin (DOX) either alone or in combination with oligonucleotides on the viability of (a) MCF-7 and (b) MDA-MB-231 breast cancer cells. The results are presented as mean ± SD, relative to un-transfected controls. * P<0.05, ** P<0.01, *** P <0.001.
Up-regulation of miR-154 promoted apoptosis in breast cancer cells
The results of flow cytometry analysis revealed that transfection with the mimic of miR-154 significantly induced apoptosis in MCF-7 and MDA-MB-231 cells (both P<0.001). On the contrary, down-regulation of miR-154 by its inhibitor decreased cell death percentage in MCF-7 and MDA-MB-231 cells (both P<0.001) (Figure 8).
Cell apoptosis assay using Annexin V and propidium iodide. A quadrant dot plot of the results of flow cytometry assay after Annexin V/PI staining and average percentage of apoptotic cells in (a) MCF-7 and (b) MDA-MB-231 cells treated with miR-154 mimic or inhibitor. The apoptotic cells were exhibited as the blue dots in the lower right quadrant of each diagram. The diagram showing the quantification of the percentage of apoptotic cells in MCF-7 (c) and MDA-MB-231 cells (d). The obtained results were compared to the untransfected controls and are presented as mean±SD. *** P <0.001.
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 confirm 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 significant increase in luciferase activity (P <0.05) (Figure 9). None of the controls significantly 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. Firefly 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.