The expression levels of miR-154 and NAMPT in breast cancer cell lines
Figure 1a shows the relative expression of miR-154 in untreated MCF-7 and MDA-MB-231 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 MCF-7 and MDA-MB-231 (both P<0.01) cell lines compared with MCF-10A cells. In addition, measurement of the NAMPT mRNA expression indicated that the expression of NAMPT was significantly higher in MCF-7 (P<0.01) and MDA-MB-231 (P<0.05) cell lines compared to MCF-10A (Figure 1b). Furthermore, the blotting results revealed that the basal level of NAMPT protein in MCF-7 and MDA-MB-231 cell lines was higher than MCF-10A (P<0.01 and P<0.05, respectively) (Figure 1 c, d).
Basal expression level of miR-154 and NAMPT in MCF-7 & MDA-MB-231 cell lines, basal level of NAMPT protein in MCF-7 & MDA-MB-231 cell lines. Basal expression of (a) miR-154 and (b) NAMPT in MCF-7 and MDA-MB-231 cell lines, compared with MCF-10A, each vertical bar represents the mean ± SD of triplicate determinations. *P<0.05; **P<0.01. (c) Evaluation of NAMPT basal expression at translational level by immunoblotting in MCF-7 and MDA-MB-231. (d) The basal protein expression levels of NAMPT in MCF-7 and MDA-MB-231 cells compared to MCF-10A cells. Each column represents the mean ± SD. *P<0.05; **P<0.01.
miR-154 cellular levels was up-regulated via miRNA mimic transfection
In order to understand the mechanism by which miR-154 controls NAMPT expression in breast cancer cells, transfection with miR-154 mimic and miR-154 inhibitor was conducted. The expression level of miR-154 was also measured after tansfection of cells with either miR-154 mimic or its antisense oligonucleotide serving as miR-154 inhibitor. The MCF-7 cell line transfected with miR-154 mimic exhibited 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). Meanwhile, the MDA-MB-231 cell line displayed a significantly enhanced cellular levels of miR-154 after transfection with miRNA-mimic (P<0.001). In contrast, transfection with miR-154 inhibitor was associated with a remarkable decrease in miR-154 level in MDA-MB-231 cells (P<0.001) (Figure 2b). Fluorescence microscopy results of the cells transfected with FAM-labeled microRNAs confirmed successful transfection (Supplementary figure 1, Additional file 1).
Relative expression of miR-154 after transfection in MCF-7 & MDA-MB-231. miR-154 cellular level after transfection of (a) MCF-7 and (b) MDA-MB-231 cells with miR-154 mimic, inhibitor or their negative controls (NC), compared to untreated 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 a target for miR-154. Subsequently, 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 human breast cancer cells (HBC) transfected with mimic, inhibitor, and their corresponding NCs. At the mRNA level, NAMPT gene revealed a significantly reduced expression in MCF-7 and MDA-MB-231 cell (both 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 NAMPT mRNA expression in both MCF-7 and MDA-MB-231 cell lines (P<0.001 and P<0.05, respectively) (Figure 3 a, b).
NAMPT gene expression in MCF-7 & MDA-MB-231 cell lines after treatment. Relative NAMPT mRNA expression in (a) MCF-7 and (b) MDA-MB-231 cells transfected with miR-154 mimic, inhibitor, their negative controls (NC) and mock 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 cell lines were transfected with miR-154 mimic and miR-154 inhibitor and western blotting was performed to evaluate whether miR-154 interfered with the regulation of NAMPT protein expression. The obtained results indicated that the up-regulation of miR-154 via transfection with miR-154 mimic, remarkably decreased the levels of NAMPT protein in both MCF-7 (P<0.05) and MDA-MB-231 (P<0.05) cells (Figure 4a, 4b). However, 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. Representative immunoblot image showing the increased basal NAMPT protein levels in MCF-7 and MDA-MB-231 compared to MCF-10A cells. Quantitation of NAMPT protein level in (a) MCF-7 and (b) MDA-MB-231 cells transfected with miR-154 mimic, inhibitor or their negative controls (NC) compared to untreated controls. Graphs represent the mean± SD of the results of the densitometric analysis of the blotting images normalized to GAPDH as the internal control and expressed relative to control cells. *P<0.05, **P<0.01, *** P <0.001.
The effect of miR-154 on NAMPT-induced NAD depletion
Increased NAMPT level is associated with high concentration of NAD in cancer cells (5). Our results showed that NAD level was decreased in the MCF-7 cells transfected with miR-154 mimic compared to un-transfected control cells (P<0.001). Moreover, NAD level was increased in the cells transfected with miR-154 inhibitor (P<0.05) (Figure 5a). Similarly, the NAD levels in MDA-MB-231 cells transfected with miR-154 mimic exhibited a significant increase (P<0.01), while a considerable decrease was observed in those transfected with miR-154 inhibitor (P<0.05) (Figure 5b).
The effect of miR-154 on intracellular NAD levels. Transfection with miR-154 mimic induced intracellular NAD depletion and reduction in cell viability. Evaluation of relative NAD levels in (a) MCF-7 and (b) MDA-MB-231 cells transfected with miR-154 mimic, inhibitor or their negative controls (NC) compared to un-transfected control. Results are presented as mean ± SD from three separate duplicate experiments.*P<0.05, ** P<0.01, ***P<0.001.
Over-expression of miR-154 in breast cancer cells reduced cell viability
NAMPT is elevated in a variety of human malignancy types including breast cancer. This enzyme induces proliferation and survival in cancer cells (19). 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) cell lines compared with the untreated cells. Whereas, treating the cells with miR-154 inhibitor significantly increased 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 presented 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 MCF-7 and MDA-MB-231 cells with doxorubicin after transfection with miR-154 mimic, miR-154 inhibitor or their negative controls. As it is shown in figure 7, miR-154 mimic synergistically decreased viability when used in combination with doxorubicin and the cell viability was significantly lower compared to either doxorubicin or miR-154 mimic alone. This effect was observed in both MCF-7 and MDA-MB-231 (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 DOX either alone or in combination 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 miR-154 mimic 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 rate in MCF-7 and MDA-MB-231 cells (both P<0.001) (Figure 8).
Cell apoptosis assay using Annexin V and propidium iodide. Representative quadrant dot plot of the flowcytometric analysis of apoptosis in breast cancer cells. Representative quadrant dot plot of Annexin V/PI staining and average percentage of apoptotic cells in (a) MCF-7 and (b) MDA-MB-231 cells transfected with miR-154 mimic, inhibitor or their negative controls. Lower-right region of each plot is indicative of the population of apoptotic cells. The blue dots in lower right quadrant of each diagram indicate apoptotic cells. (c) and (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 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 containing NAMPT-related 3′-UTR in the presence of miR-154 mimic, inhibitor or their negative controls 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 miR-154 mimic, inhibitor, their negative controls and mock into HEK293T. Firefly luciferase activity was normalized with respect to renilla luciferase as control. Results are shown as mean±SD of three independent experiments. * P<0.05, ** P <0.01.