Correction: MiRNA-26b inhibits proliferation by targeting PTGS2 in breast cancer

After publication of this article [1] , the authors noted an error in Figure 1. The labelling of normal tissues and cancer tissues was inverted. Please see figure 1 for the corrected version. The authors apologise for any inconvenience this has caused.


Introduction
MicroRNAs are a class of small, non-coding RNAs, which are capable of regulating gene expression at the posttranscriptional level. Mechanistically, miRNAs function by binding to the 3 0 untranslated regions (UTRs) of target mRNAs, causing translation to be blocked and/or mRNA degradation [1]. MicroRNAs play diverse roles in tumorigenesis and in the progression of breast cancer, and may act as oncogenes, tumor suppressors and modulators of tumor proliferation, invasion, apoptosis and therapy resistance [2][3][4][5][6]. An increasing body of evidence indicates that miR-26b is downregulated in hepatocellular carcinoma [7], nasopharyngeal carcinoma [8], primary squamous cell lung carcinoma [9], squamous cell carcinoma of the tongue [10] and in breast cancer [11]. Furthermore, overexpression of miR-26b induces apoptosis in MCF-7 breast cancer cells by targeting SLC7A11 [11]. However, to date, the role of miR-26b in breast cancer tumorigenesis is incompletely understood.
Prostaglandin-endoperoxide synthase-2 (PTGS2) encodes the COX-2 enzyme, which catalyzes the conversion of arachidonic acid to prostaglandins (PGs) and other eicosanoids. PTGS2 expression, which is undetectable in most normal tissues, is induced in response to hypoxia, inflammatory cytokines, tumor promoters, growth factors and other stressors [12,13]. PTGS2 is involved in carcinogenesis, immune response suppression, inhibition of apoptosis, angiogenesis and tumor cell invasion and metastasis. Recent studies have indicated that PTGS2 genetic variation is associated with breast cancer susceptibility [14,15]. Furthermore, overexpression of PTGS2 in patients with breast cancer is associated with a worse prognosis [16]. Several studies have demonstrated the involvement of miRNAs in the regulation of PTGS2. MiR-199a and miR-101a are implicated in PTGS2 regulation during embryo implantation [17]. Furthermore, miR-26b has been shown to directly silence PTGS2 and regulate PTGS2 expression in desferrioxamine (DFOM)-treated carcinoma of nasopharyngeal epithelial (CNE) cells [8].
In this study, we report that miR-26b expression is significantly decreased in human breast cancer, and its overexpression inhibits the proliferation of MDA-MB-231 cells by targeting PTGS2. These results indicate that miR-26b functions as a tumor suppressor, whose dysregulation may be involved in the initiation and development of human breast cancer.

Expression of miR-26b is decreased in human breast cancer
To investigate the involvement of miR-26b in breast cancer development, we analyzed levels of miR-26b in 38 invasive ductal breast cancer tissues and associated normal adjacent tissues (NATs) by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). MiR-26b expression was significantly decreased in breast cancer tissues compared with NATs (7.3 fold, P<0.01) (Figure 1).

Suppression of breast cancer proliferation by miR-26b
To investigate the effect of miR-26b on breast cancer cell proliferation, miR-26b mimics were transfected into the human breast cancer cell line, MDA-MB-231 and proliferation was assessed by MTT assay. As shown in Figure 2, cellular proliferation gradually declined following transfection with miR-26b, in a concentration-dependent manner. Treatment of cells with 50 nM miR-26b led to a decrease in MDA-MB-231 cell growth at 72 h (7%) and 96 h (18%) (P<0.05) compared with the negative control. This inhibitory effect was significantly enhanced following transfection with 100 nM miR-26b at 48 h (14%), 72 h (29%) and 96 h (28%) (P<0.05) compared with the negative control. Taken together, these results demonstrate that miR-26b inhibits the proliferation of MDA-MB-231 breast cancer cells.

MiR-26b regulates PTGS2 expression in breast cancer cells
To investigate the downstream targets of miR-26b that may play a role in mediating this growth suppressive effect, we searched for putative targets using the miRanda database. We identified a binding site for miR-26b in the 3 0 -UTR of PTGS2 mRNA. To validate miR-26b binding to this predicted site, we cloned the 3 0 -UTR of PTGS2 containing the putative miR-26b binding site into a luciferase reporter construct, in addition to a mutated PTGS2 3 0 -UTR ( Figure 3A). Luciferase activity was significantly decreased following co-transfection of psiCHECK-2/PTGS2 3 0 -UTR with miR-26b, compared with the miR-negative control (miR-NC) ( Figure 3B). Furthermore, luciferase activity was also decreased following co-transfection of psiCHECK-2 /PTGS2 3 0 -UTR mutant and miR-26b ( Figure 3B). These results indicate that miR-26b specifically binds to the 3 0 -UTR of PTGS2. The effect of miR-26b transfection on endogenous PTGS2 mRNA and protein expression was subsequently evaluated in MDA-MB-231 cells by qRT-PCR and Western blot. As shown in Figure 3C and 3D, the expression of PTGS2 mRNA and protein was decreased in MDA-MB-231 cells transfected with 100 nM miR-26b mimics compared with the control. These results suggest that miR-26b directly targets PTGS2 in breast cancer cells.

MiR-26b inhibits the proliferation of breast cancer cells via regulation of PTGS2
Since overexpression of miR-26b suppressed the proliferation of MDA-MB-231 breast cancer cells, and given that PTGS2 is a direct target of miR-26b, we hypothesized that the inhibitory effect of miR-26b on breast cancer cell viability might be achieved via targeting PTGS2. To investigate this, we assessed the effect of targeted knockdown of PTGS2 on MDA-MB-231 cell growth by MTT assay. Treatment of cells with 50 nmol/L PTGS2 siRNA markedly suppressed cell viability by 21%, 34% and 41% at 48 h, 72 h and 96 h, respectively, compared with control siRNA (P<0.01) ( Figure 4). This suggests that PTGS2 promotes the proliferation of breast cancer cells in vitro. These results demonstrate that downregulation of PTGS2 expression by miR-26b contributes, at least in part, to the suppression of the growth of breast cancer cells.

Discussion
The discovery of the first miRNA, lin-4, in Caenorhabditis elegans initiated a new era of miRNA biology. Since then, thousands of miRNAs have been identified and annotated. Furthermore, an increasing body of evidence indicates that miRNAs are differentially expressed between normal and tumor tissues, suggesting that dysregulation of miRNA  expression is a key factor underlying tumorigenesis [18][19][20][21][22]. In this study, we performed qRT-PCR to investigate the expression pattern of miR-26b in primary human breast cancer. MiR-26b expression was significantly downregulated in breast cancer specimens compared with normal tissue. Similar findings have been reported in several other cancer types, including hepatocellular carcinoma [7], nasopharyngeal carcinoma [8], primary squamous cell lung carcinoma [9], squamous cell carcinoma of tongue [10] and glioma [23]. Consistent with this study, Xiao-Xiao Liu et al. reported that miR-26b expression is downregulated in MCF7, HCC1937, MDA-MB-231, MDA-MB-468, MDA-MB-453, BT-549 and BT-474 breast cancer cell lines compared with CCD-1095Sk normal breast skin cells [11]. These results indicate that miR-26b is downregulated in human breast cancer specimens and cell lines.
It is well established that regulation of gene expression by miRNAs plays a role in the development, differentiation, proliferation, apoptosis, invasion and metastasis of a variety of cancers. In our study, transfection of miR-26b mimics into MDA-MB-231 cells led to a significant decrease in cellular proliferation, indicating that miR-26b represses the growth of breast cancer cells. Previous studies demonstrated that overexpression of miR-26b in DFOMtreated CNE cells inhibited proliferation via degradation of PTGS2 mRNA and suppression of PTGS2 protein translation [8]. Gain-and loss-of-function studies showed that miR-26b and its host genes CTDSP1/2/L cooperate to block G1/S-phase progression by activating pRb protein in hepatocellular carcinoma [24]. MiR-26b is also involved in processes governing apoptosis in breast cancer. Previous studies reported that miR-26b mimics triggered apoptosis of human breast cancer MCF7 cells, and SLC7A11 was identified as a direct target of miR-26b [11]. In glioma, low levels of miR-26b were inversely correlated with tumor grade. Ectopic expression of miR-26b inhibited the proliferation, migration and invasion of human glioma cells, possibly via regulation of its downstream target, EphA2 [23]. Taken together, these studies indicate that dysregulated expression of miR-26b may affect multiple cancers.
Computational algorithms revealed that the 3 0 -UTR of PTGS2 contains a binding site for miR-26b. To confirm targeting of PTGS2 by miR-26b, we integrated a fragment of the PTGS2 3 0 -UTR containing the target sequence, or a fragment whose target site was mutated, into a luciferase reporter vector. Luciferase activity was significantly repressed in cells transfected with the construct harboring the miR-26b target sequence compared with the mutated control vector. Both PTGS2 mRNA and protein levels decreased after transfection of MDA-MB-231 cells with miR-26b, as shown in Figure 3C and 3D. These data indicate that miR-26b directly interacts with PTGS2 mRNA and represses PTGS2 protein expression. Furthermore, silencing PTGS2 expression by siRNA led to inhibition of cellular proliferation. In conclusion, these findings support the hypothesis that decreased expression of PTGS2 by miR-26b accounts for the suppression of cellular proliferation in breast cancer.

Conclusions
Taken together, we demonstrate that miR-26b is downregulated in breast cancer specimens compared with normal tissue. MiR-26b directly downregulates PTGS2 and inhibits breast cancer cell proliferation. These data indicate that miR-26b may serve as a tumor suppressor gene involved in breast cancer pathogenesis.

Specimens
In this study, 38 paired breast cancer and normal specimens were collected from the Department of Breast and Thyroid Surgery of Shanghai Tenth People's Hospital, Shanghai, China. All samples were confirmed as invasive, ductal breast cancer by trained pathologists. No patients received chemotherapy or radiotherapy prior to surgery.

Western blot analysis
Protein samples were separated by 12% SDSpolyacrylamide gel (SDS-PAGE) and transferred onto PVDF membranes (Beyotime, China). Immune complexes were formed by incubation of membranes with primary antibody (BioVision, USA) overnight at 4°C. Blots were washed and incubated for 1 h with HRP-conjugated antirabbit secondary antibody. Immunoreactive protein bands were detected using an Odyssey Scanning system.

MTT assay
MDA-MB-231 cells (5000/well) were plated in 96-well plates (BD Biosciences, USA) and incubated at 37°C overnight. The next day, sub-confluent (50-60%) cells were transfected with miR-26b mimics (50 nmol/L and 100 nmol/L) or PTGS2 siRNA (50 nmol/L) (Genepharma, China) using Lipofectamine 2000 (Invitrogen, USA), in accordance with the manufacturer's instructions. MiR-NC and siRNA control were used as negative controls. DMEM medium was replaced with DMEM supplemented with 10% FBS 5 h post-transfection with miR-26b mimics or PTGS2 siRNA. Cell proliferation was assessed at 24, 48, 72 and 96 h, using the MTT proliferation assay kit in accordance with the manufacturer's instructions (Sigma, USA). All experiments were performed in biological triplicate.

Statistical analysis
Data are presented as the mean ± standard deviation from at least three independent experiments. The twotailed t-test was used to draw a comparison between groups. The null hypothesis was rejected at the 0.05 level.