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. 2015 Jul 1;8(7):7729-39.
eCollection 2015.

MicroRNA-497 induces cell apoptosis by negatively regulating Bcl-2 protein expression at the posttranscriptional level in human breast cancer

Chuankui Wei  1 , Qifeng Luo  2 , Xiaoguo Sun  3 , Dengfeng Li  2 , Hongming Song  2 , Xiaoyu Li  2 , Jialu Song  2 , Kaiyao Hua  2 , Lin Fang  2
Affiliations

MicroRNA-497 induces cell apoptosis by negatively regulating Bcl-2 protein expression at the posttranscriptional level in human breast cancer

Chuankui Wei et al. Int J Clin Exp Pathol. .

Abstract

Many studies have demonstrated that microRNAs (miRNAs) may play vital roles in the development of breast cancer. The aim of this study was to examine the expression levels of miR-497 in human breast cancer and investigate whether its potential roles involved targeting Bcl-2. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to examine the expression levels of miR-497 in 48 breast cancer specimens and six breast cancer cell lines. MTT assay, colony formation assay, and flow cytometry were conducted to explore the potential functions of miR-497 in human MDA-MB-231 breast cancer cells. Correlation analysis and dual-luciferase reporter assay were performed to validate whether Bcl-2 was a direct target of miR-497. The effects of modulating miR-497 on endogenous levels of Bcl-2 were subsequently confirmed via qRT-PCR and western blot. MTT assay, colony formation assay and flow cytometry were used to indicate the roles of endogenous Bcl-2 in breast cancer cells. miR-497 expression levels were significantly decreased in human breast cancer specimens and cell lines (P<0.05). Overexpression of miR-497 in breast cancer cells suppressed cell proliferation and induced apoptosis. Correlation analysis indicated that miR-497 was highly inversely correlated with Bcl-2 protein expression in breast cancer specimens. Dual-luciferase reporter assays confirmed that Bcl-2 was a direct target of miR-497. qRT-PCR and western blot showed that miR-497 negatively regulated Bcl-2 protein expression but had no impact on mRNA expression of Bcl-2. Knockdown of Bcl-2 expression in MDA-MB-231 cells significantly suppressed cell proliferation and promoted apoptosis. Our study suggests that miR-497 may act as a breast cancer suppressor through negative regulation of Bcl-2 protein expression at the posttranscriptional levels. Therefore, targeting miR-497 may provide a novel strategy for the diagnosis and treatment of patients with this lethal disease.

Keywords: Bcl-2; apoptosis; breast cancer; miR-497.

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Figures

Figure 1
Figure 1
miR-497 is downregulated in human breast cancer cell lines and clinical specimens. A. Relative expression of miR-497 was downregulated in breast cancer cell lines compared to MCF-10A cells. B. Relative miR-497 expression in 48 paired breast cancer tissues was downregulated compared to matched adjacent normal breast tissues. Data represent the 2-ΔΔCt values ± SD,*P<0.05.
Figure 2
Figure 2
Overexpression of miR-497 repressed MDA-MB-231 cell proliferation. A. Relative miR-497 expression of miR-NC group were lower than miR-497 mimics group and higher than miR-497 inhibitor group. B. Upregulation of miR-497 significantly repressed the growth rate of MDA-MB-231 cells, while the inhibitor increased the growth rate (P<0.05). C. Dose-dependent effect of miR-497 mimics on cell growth. The optical density of each well was measured at the indicated time-points with a microplate reader at 490 nm. 100 nM was selected as the optimal concentration for subsequent analyses. The graph represents OD 490 nm ± SD, P<0.05.
Figure 3
Figure 3
Overexpression of miR-497 inhibited colony formation ability of MDA-MB-231 cells. A. Crystal violet stained colonies from the colony formation assay for the miR-497 mimics group, miRNA NC group, and miR-497 inhibitor group. B. Cells transfected with miR-497 mimics exhibited fewer colonies than the NC group; cells transfected with miR-497 inhibitor exhibited higher numbers of colonies than the NC group (P<0.05).
Figure 4
Figure 4
miR-497 mimics significantly induced early and late apoptosis of breast cancer cells. In contrast, cells transfected with negative control and inhibitor exhibited low levels of apoptosis (P<0.05).
Figure 5
Figure 5
Bcl-2 protein expression was inversely correlated with miR-497 miRNA expression in breast cancer specimens. Normalized Bcl-2 protein expression (to β-actin) is shown compared to miR-497 levels by qRT-PCR. ACT: β-actin.
Figure 6
Figure 6
Bcl-2 is a direct target of miR-497. A. The miR-497 binding site in the 3’-UTR of Bcl-2 mRNA and the Bcl-2 3’-UTR mutant sequence. B. The relative luciferase activity (Firefly/Renilla) was measured in HEK293T cells after co-transfection of the Bcl-2 3’-UTR luciferase construct with miR-497 mimics or miR-NC and co-transfection of the Bcl-2 3’-UTR mutant luciferase construct with miR-497 mimics or miR-NC.
Figure 7
Figure 7
miR-497 negatively regulated Bcl-2 protein expression at the posttranscriptional level. A. No differences were detected in the levels of Bcl-2 mRNA between miR-497 mimics, inhibitor and negative control cells, P>0.05. B. Cells transfected with miR-497 mimic showed decreased endogenous Bcl-2 protein compared with NC. miR-497 inhibitor showed enhanced Bcl-2 protein expression compared with NC.
Figure 8
Figure 8
Effects of Bcl-2 in breast cancer cell proliferation and apoptosis. A. Cells transfected with Bcl-2 siRNA showed markedly inhibited cell proliferation compared with control siRNA (P<0.05). B, C. Cells transfected with Bcl-2 siRNA exhibited fewer colonies than the siRNA NC group (P<0.05). D. Inhibition of Bcl-2 promoted the ability of cells to undergo apoptosis (P<0.05). E. The level of Bcl-2 protein was decreased in the Bcl-2 siRNA group compared with siRNA NC group.
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