Microrna-124 targets flotillin-1 to regulate proliferation and migration in breast cancer

Background MicroRNAs (miRNAs) have been documented as playing important roles in cancer development. In this study, we investigated the role of miR-124 in breast cancer and clarified the regulation of flotillin-1 (FLOT1) by miR-124. Methods The expression levels of miR-124 were examined in breast cancer cell lines and patient specimens using quantitative reverse transcription-PCR. The clinicopathological significance of the resultant data was later analyzed. Next, we explored the function of miR-124 to determine its potential roles on cancer cell growth and migration in vitro. A luciferase reporter assay was conducted to confirm the target gene of miR-124, and the results were validated in cell lines and patient specimens. Results We found that miR-124 expression was significantly downregulated in breast cancer cell lines and patient specimen compared with normal cell lines and paired adjacent normal tissues (P < 0.0001), respectively. MiR-124 was also associated with tumor node metastasis (TNM) stage (P = 0.0007) and lymph node metastasis (P = 0.0004). In breast cancer cell lines, the ectopic expression of miR-124 inhibited cell growth and migration in vitro. Moreover, we identified the FLOT1 gene as a novel direct target of miR-124, and miR-124 ectopic expression significantly inhibited FLOT1. Luciferase assays confirmed that miR-124 could directly bind to the 3′ untranslated region of FLOT1 and suppress translation. Moreover, FLOT1 was widely upregulated, and inversely correlated with miR-124 in breast cancer tissues. Consistent with the effect of miR-124, the knockdown of FLOT1 significantly inhibited breast cancer cell growth and migration. We also observed that the rescue expression of FLOT1 partially restored the effects of miR-124. Conclusions Our study demonstrated that miR-124 might be a tumor suppressor in breast cancer via the regulation of FLOT1. This microRNA could serve as a potential diagnostic marker and therapeutic target for breast cancer.


Background
Breast cancer is the leading cause of cancer death in females worldwide. Due to the advances in diagnosis and appropriately systemic therapy, including surgery, radiation and chemotherapy, the prognosis of breast cancer is encouraging. However, similar to many other solid tumors, distant metastases account for more than 90% of breast cancer-related death [1]. Because the underlying mechanisms of breast cancer metastasis consist of multiple sequential steps that are not completely understood to date, further investigation of this mechanism is urgently needed.
MicroRNAs (miRNAs) are endogenous noncoding small RNAs that contribute to the regulation of their cognate target genes by usually imperfect base-pairing to the 3′ untranslated region (UTR) of a target mRNA, which results in either mRNA degradation or translation inhibition [2]. In fact, miRNAs are implicated in the regulation of various cellular processes, including proliferation, differentiation, cell death and cell mobility [3]. Furthermore, miRNA profiles also indicate that miRNAs can function either as oncogenes or tumor suppressors in tumor progression [4,5]. Therefore, miRNA expression profiles constitute progress in cancer diagnosis, classification, clinical prognostic information and therapy [6][7][8][9][10].
FLOT1 is overexpressed in several types of cancer, including breast cancer [23][24][25][26]. FLOT1 was originally identified as a marker of lipids, which is important for non-caveolar raft formation and associated with the development and progression of cancer. In breast cancer, the FLOT1 expression level correlated with clinical staging and prognosis, and its silencing inhibited the proliferation and tumorigenicity of breast cancer cells in vitro and vivo [26]. MicroRNAs can regulate the expression levels of FLOT1 [27], a process that was intensively studied by our group. Our findings, consistent with other groups, indicated that the role of miR-124 in the growth and metastasis inhibition was accomplished by the regulation of FLOT1 in breast cancer.
In this study, we aimed to investigate the role of miR-124 in breast cancer. We found that downregulation of miR-124 in breast cancer tissues compared with the corresponding normal tissues, and inversely associated with TNM stage and lymph node metastasis in breast cancer. In addition, synthetic miR-124 mimics inhibited the growth and migration of breast cancer cells in vitro. Furthermore, we validated FLOT1, which was overexpressed in breast cancer and predicted as the target of miR-124, by 3′-UTR luciferase assays and western blot analysis. Finally, knockdown of FLOT1 consistent with the effects of miR-124 in breast cancer, and rescue expression of FLOT1 could partially restore these miR-124 effects. Our study demonstrated that miR-124 acts as a tumor suppressor by directly targeting FLOT1 in breast cancer, which suggested that miR-124 has potential diagnostic and therapeutic value for breast cancer treatment.

Results
MiR-124 was downregulated in breast cancer cell lines and clinical specimens and inversely associated with advanced clinical stage and lymph node metastasis To study the expression level of miR-124 in breast cancer, a panel of breast cancer cell lines was first analyzed by stem-loop RT-PCR. Compared with the two immortalized normal mammary epithelial cell lines (184A1, MCF-10A), miR-124 expression level was downregulated in all 7 breast cancer cell lines (MDA-MB-231, MDA-MB-361, MDA-MB-435, MDA-MB-468, MCF-7, HBL100, T47D, 4 T1) ( Figure 1A).
We further assessed the expression levels of miR-124 in 78 clinical human primary breast cancer tissues and 40 paired normal adjacent tissues (NATs) to analyze the clinicopathologic significance of the miR-124. The relationship between the miR-124 expression levels and clinicopathologic characteristics in breast cancer patients are summarized in Table 1. Consistent with the result obtained from breast cancer cell lines, the average expression level of miR-124 was downregulated in breast cancer tissues compared with paired normal adjacent tissues ( Figure 1B; P < 0.0001). We divided 78 breast cancer cases into two groups according to the status of lymph node metastasis: lymphatic node metastasis positive or negative. Interestingly, the breast cancer lymphatic node metastasis positive group (n = 37) showed an even lower miR-124 expression level than the lymphatic node metastasis negative group (n = 41; Figure 1C; P = 0.0004). In addition, we also found that the expression of miR-124 was lower in advanced TNM stage breast cancer patients (stage III and IV) than early stage patients (stage I and II; Figure 1D and Table 1). Taken together, these results indicated that miR-124 is downregulated in breast cancer, and a reduced expression of miR-124 may play an important role in the progression and metastasis of breast cancer.

Ectopic expression of miR-124 inhibited the proliferation, migration and invasion of breast cancer cells
To investigate the effect of miR-124 on cell proliferation, we transfected the breast cancer cell lines MDA-MB-231 and T47D with miR-124 mimics. The successful overexpression of miR-124 in the cells was confirmed by quantitative real-time PCR (Figure 2A). MTT and colony formation assays showed that ectopic expression of miR-124 could markedly inhibit the proliferation and growth of MDA-MB-231 and T47D cells compared with the mimic control ( Figure 2B and D; P < 0.05). This antiproliferation effect could be partially due to the disruption of cell growth regulation, such as cell cycle arrest. Thus, we next explored the effect of miR-124 on cell cycle regulation. Flow cytometric cell cycle analysis showed that miR-124 increased the number of cells in the G0 + G1 phase and decreased the number of cells in the S and G2 + M phase in the MDA-MB-231 and T47D breast cancer cell lines compared with miR-Ctrl ( Figure 2C).
After confirming the cell proliferation and growth inhibition ability of miR-124, we investigated the role of miR-124 in cell migration and invasion. Wound healing and Matrigel invasion assays demonstrated that the ectopic expression of miR-124 inhibited the cell migration and invasion of MDA-MB-231 and T47D cells compared with the mimic control ( Figure 2E and F). The above results support the role of miR-124 in the inhibition of breast cancer proliferation, migration and invasion and suggest that miR-124 has a tumor suppressor function.

MiR-124 downregulated FLOT1 expression by directly targeting its 3′-UTR
To elucidate the molecular mechanism responsible for the proliferation and migration inhibition induced by miR-124 in breast cancer cells, we used a bioinformatic analysis to search for putative protein-coding gene targets of miR-124, especially for those that can promote cancer cell growth and metastasis. According to this rationale, FLOT1 was selected as one of the candidate targets of miR-124, which was highly conserved among different species and whose 3′-UTR of mRNA contained a complementary site for the seed region of miR-124 ( Figure 3A).
We performed a luciferase reporter assay to determine whether FLOT1 is a direct target of miR-124 in breast cancer cells. The target region sequence of FLOT1 3′-UTR (wt 3′-UTR) or the mutant sequence (mut 3′-UTR) was cloned into a luciferase reporter vector ( Figure 3B). These constructed reporter vectors were co-transfected with miR-124 mimics or miR-Ctrl into the MDA-MB-231 cell line. The data in Figure 3C show that miR-124 could downregulate the luciferase activity of the FLOT1 wt 3′-UTR construct ( Figure 3C, lanes 3; P < 0.05), whereas the luciferase activity was not significantly attenuated in the target region of the mutated mut 3′-UTR construct ( Figure 3C, lanes 4, 5 and 6). These data suggest that the regulation of miR-124 on FLOT1 depended on the specific seed region sequence. Moreover, miR-Ctrl did not significantly affect the luciferase activity of either the wt or mut 3′-UTR construct ( Figure 3C, lanes 2 and 5).
We further analyzed the FLOT1 protein expression by using western blotting after transfecting MDA-MB-231 and T47D cells with miR-124 mimics. As shown in  Figure 3D, the ectopic expression of miR-124 inhibited FLOT1 expression by approximately 60% to 70%. Therefore, we concluded that miR-124 inhibited FLOT1 expression by binding to the 3′-UTR sequences of FLOT1 in breast cancer.

Knockdown of FLOT1 induced inhibition of breast cancer cells proliferation and invasion
To explore the function of FLOT1 in breast cancer, MDA-MB-231 and T47D cells were transfected with FLOT1-specific siRNAs (FLOT1-siRNA). A western blot analysis indicated that FLOT1 protein decreased significantly after 48 hours in both MDA-MB-231 and T47D cells transfected with 50 nM FLOT1-siRNA ( Figure 4A). The MTT and colony formation assays showed that the knockdown of FLOT1 inhibited the proliferation and growth of both MDA-MB-231 and T47D cells ( Figure 4B; P < 0.05). Furthermore, a Matrigel invasion assay indicated that the knockdown of FLOT1 inhibited the invasion of breast cancer cells ( Figure 4C), an effect that resembled the inhibitory effect of miR-124 in breast cancer cells.
To test whether FLOT1 is the direct functional mediator of the miR-124 induced inhibition of breast cancer cell proliferation and migration, we co-transfected miR-124 mimics along with wt/mut 3′-UTR-FLOT1 plasmid which FLOT1 cDNA contained wild type or mutant 3′ UTR into breast cancer cells. MTT and Matrigel invasion assays showed that mut 3′-UTR-FLOT1 could partially abrogate the miR-124-mediated effects in breast cancer cells, to restore the proliferation and migration compared with the miR-Ctrl ( Figure 4D and E; P < 0.05). Therefore, FLOT1 has an important role in the proliferation and invasion of breast cancer cells, which was regulated by miR-124.
MiR-124 and FLOT1 are inversely correlated in breast cancer tissues IHC was performed to detect FLOT1 expression in 5 normal breast tissues and 78 clinical breast cancer specimens, and the miR-124 expression levels were simultaneously analyzed by RT-PCR. These cases included 21 cases of clinical stage I, 41 cases of stage II, 13 cases of stage III, and 3 cases of stage IV breast cancers. FLOT1 was found to be predominantly overexpressed in the cytoplasm and membranes of breast cancer tumor cells and was less expressed in adjacent normal tissues ( Figure 5A). In consensus with previous report [26], our data also showed that FLOT1 expression in stage I to IV primary tumors was statistically higher than in normal breast tissues (P < 0.05, Figure 5B). We then correlated FLOT1 with the miR-124 expression levels in the same breast cancer specimens. As shown in Figure 5C, a significant inverse correlation was observed when FLOT1 expression levels were plotted against miR-124 expression levels (2-tailed Spearman's correlation, r = −07437; P <0.0001).

Discussion
Cancer is characterized by abnormal and uncontrolled cell proliferation, which is caused not only by the misregulation of several pivotal proteins but also by a systemic change in the miRNAs profile [28]. MiRNAs are involved in the regulation of multiple biological processes, including development, cell proliferation, apoptosis, differentiation, disease survival and cell death [29,30]. Considering the function of miRNAs, their deregulation expectedly contributes to substantial cell physiological and pathological processes and is ultimately involved in tumorigenesis and the tumor progression of many different human cancers. In this report, we showed that miR-124 was markedly downregulated in human breast cancer cell lines and clinical specimens compared with immortalized normal mammary epithelial cell lines and normal adjacent tissues, respectively. MiR-124 downregulation was significantly associated with advanced clinical stage and positive lymph node-metastasis in breast cancer patients. Furthermore, the ectopic expression of miR-124 inhibited breast cancer cell proliferation, migration and invasion. Moreover, FLOT1 was identified as a direct and functional target of miR-124 via binding to the 3′ UTR of FLOT1. Our study suggested that miR-124 acts as a novel proliferation and metastasis suppressor in breast cancer, and downregulated miR-124 contributes to lymph node-metastasis and tumor progression in breast cancer patients.
Although miR-124 was identified long ago, its biological function has only recently been investigated. MiR-124 acts as a tumor suppressor, and its downregulation has been identified in various cancers [16,[18][19][20][21][22][31][32][33], which suggests that miR-124 may play a vital role in tumorigenesis and tumor progression. Shi et al. showed that miR-124 was a potential tumor-suppressive miRNA and was downregulated in prostate cancer to result in proliferation inhibition of prostate cancer cells by targeting the androgen receptor [20]. Wang et al. reported that miR-124 was epigenetically silenced in pancreatic cancer and inhibited cell proliferation and metastasis by regulating Rac1 [17]. Zheng et al. showed that miR-124 levels were frequently reduced in hepatocellular carcinoma, and this expression level was significantly associated with the patients' clinical stages and prognoses and regulated the invasion and migration of hepatocellular carcinoma through post-transcriptional regulation of ROCK2 and EZH2 [19].  the ectopic expression of miR-124 could suppress the invasion and metastatic ability, likely by directly targeting the CD151. CD151 regulates the ligand biding activity of integrin α3β1 and plays a role in Met-dependent signaling and TGF-β signaling, while c-met can regulate many cellular process, especially the proliferation and migration of cancer. These results suggest an important role for miR-124 in the proliferation and metastasis of different cancers. However, the miR-124 expression levels in clinical specimens and its exact mechanism in breast cancer has not been clearly elucidated. In this study, we demonstrated for the first time that miR-124 was frequently downregulated in breast cancer, and the average expression levels of miR-124 were significantly downregulated in breast cancer tissues compared with paired normal adjacent tissues. Interestingly, we found that lower levels of miR-124 are associated with advanced TNM stage (stage I + II vs. stage III + IV, P = 0.0007) and positive lymph node metastasis, suggesting that a low expression of miR-124 is associated with breast cancer progression. Recently, miR-124 was reported to be subject to epigenetic regulation in various tumors, including breast cancer [17,31,32,34], which in turn may explain the downregulation of miR-124 in breast cancer. Taken together, these results suggest that miR-124 expression is frequently reduced in breast cancer, which may be responsible for the tumorigenesis and progression of breast cancer. However, the function of miR-124 in breast cancer is not fully understood.
The capability of cells to proliferate, migrate and invade is considered an important determinant in the process of tumorigenesis and progression. Many oncogenes and suppressor genes reportedly correlate with the course of cancer initiation and progression, but the molecular mechanisms are not fully understood. Recently, accumulating studies have reported that miRNAs play important roles in breast cancer tumorigenesis and progression [35][36][37]. Interestingly, a number of miRNAs are associated with the proliferation and migration of breast  cancer, such as miR-26a [38], miR-34a [39], miR-137 [40], and miR-210 [41], which may provide new insights into the design of eradicating therapeutic strategies for breast cancer. Even miR-124 has been reported as a tumor suppressor miRNA in breast cancer. However, the mechanisms involved have not been fully elucidated. To determine the function of miR-124 in breast cancer, we tested the effect of miR-124 on MDA-MB-231 and T47D cell lines. Ours results indicate that miR-124 could suppress breast cancer cell proliferation, migration and invasion, which suggests its role as a tumor suppressor in breast cancer.
In the current study, we identified FLOT1 as a direct and functional target of miR-124. The protein encoded by the FLOT1 gene is an integral membrane protein that participates in vesicular trafficking, signal transduction and is important for lipid raft formation [25,26,42]. Accumulating evidence shows that the overexpression of FLOT1 in various cancers contributes to proliferative and invasive behavior as well as a worse prognosis [24][25][26]. The knockdown of FLOT1 reportedly suppressed the proliferation and tumorigenesis of breast cancer cells by enhancing the transcriptional activity of FOXO3a, inhibiting Akt activity, downregulating cyclin D1 and upregulating the cyclin-dependent kinase inhibitors p21 Cip1 and p27 Kip1 [26]. Xiong et al. also showed that Flotillin-1 could clearly activate the growth and metastasis of oral squamous carcinoma by transfecting cells with a Flotillin-1 expression vector or shRNA targeted Flotillin-1. This effect was mediated by the activation of the NF-κB signaling pathway, which enhanced the phosphorylation of p65 and IκBα [23]. These studies showed that FLOT1 can regulate many cellular processes, particularly in cancer growth, proliferation, migration, metastasis and tumorigenesis. Consistent with the study above, we found that miR-124 could directly target and downregulate FLOT1, and high FLOT1 expression was associated with low miR-124 levels in breast cancer specimens. These findings provide new insight into the essential mechanisms of FLOT1 regulation in breast cancer. Additionally, miR-138 was also reported to regulate FLOT1 in esophageal squamous cell carcinoma. These findings suggest that the post-transcriptional regulation of FLOT1 by miRNAs is a vital mechanism underlying cancer proliferation and metastasis, and miR-124 may serve as potential treatment target for regulating FLOT1 to inhibit the growth and metastasis of breast cancer.

Conclusions
Our study demonstrates that miR-124 is downregulated and inversely associated with the lymph node metastasis in breast cancer. The ectopic expression of miR-124 inhibits cell proliferation and migration by downregulating FLOT1, which indicates the internal mechanism of tumor suppression of miR-124. Combined with the above mentioned studies, this work contributes to the understanding of the effect of miR-124 on tumor suppression. This study suggests that miR-124 downregulated may play an important role in tumor proliferation and migration and may be a novel diagnostic marker and potential therapeutic target in breast cancer.

Materials and methods
Human breast cancer tissues  Table 1. The tumor stage was defined according to the American Joint Committee on Cancer and tumorlymph node-metastasis classification system [43]. All patients provided consent for the use of their specimens in research, and this use was approved by the institute research ethics committee of the First Hospital of Sun Yat-sen University.

Immunohistochemical staining
Inmmunohistochemistry (IHC) staining of formalinfixed and paraffin-embedded tissue slides was performed and quantified as previously described [44]. Briefly, 5 μm tissue slides were deparaffinized, rehydrated via a series of descending graded alcohols and subjected to antigen retrieval in 0.01 M citrate buffer (pH 6.0) at 90°C for 40 minutes. Following a blocking step, the slides were incubated with FLOT1 primary antibody (1:500; Sigma, Saint Louis, MO) and washed. Biotinylated secondary antibody was applied, and the immunocomplexes were visualized using an avidin-biotin complex immunoperoxidase system (Vector Laboratories, Burlingame, CA, USA) with 0.03% diaminobenzidine (DAB) as a chromagen and hematoxylin as the counterstain. We used phosphatebuffered saline (PBS) instead of the primary antibody as a negative control, and a composite slide containing formalin-fixed cell pellets of MDA-MB-231 and T47D as positive control to assess the quality of the IHC reaction. The slides were reviewed and scored independently based on both the percentage of positive stained tumor cells and overall stained intensity by two observers who were blinded to specimens' clinical information. The following scoring rubric was used: scored 0, absent positive tumor cells; scored +, weak cell staining or <10% positive tumor cells; scored ++, moderate cell staining or 10-50% positive tumor cells; scored +++, strong cell staining or >50% positive tumor cells. Conflicts (approximately 5% of cases) were resolved by consensus.

Cell culture
The

Bioinformatics
The analysis of miR-124 predicted targets was determined using the algorithms of TargetScan 5.1 (http:// www.targetscan.org/) and miRanda (http://www.microrna.org). According to these algorithms, we predicted that the FLOT1 gene might be a direct target of miR-124.

RNA extraction and quantitative real-time PCR
The RNA extraction and quantitative real-time PCR procedure were carried out as previously reported [39]. Briefly, total RNA was extracted using TRIzol® Reagent (Invitrogen, CA, USA). To quantitate the miR-124 expression, reverse transcription was performed with a specific stem-loop real-time PCR miRNA kit (RiboBio, Guangzhou, China). Quantitative real-time PCR (qPCR) was performed using the Platinum SYBR Green qPCR SuperMix-UDG system (Invitrogen, CA, USA) on an Applied Biosystems 7900HT real-time PCR system, and the data were collected and analyzed using ABI SDS version 2.3. All procedures were performed according to the manufacturer's instructions. 5S rRNA was used as an internal control. All samples were normalized to internal controls, and the fold changes were calculated according to the relative quantification method (RQ = 2 −ΔΔCT ). The results are shown as fold changes of expression in cells or cancer tissues.

MTT assay
The cell viability and proliferation of MDA-MB-231 and T47D with miRNA mimics or siRNA duplexes were determined by 3-(4, 5-dimethylthiazolyl-2-yl)-2-5 diphenyl tetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) assay. The cells were plated in 96-well plates at 5 × 10 3 per well in a final volume of 100 μL and treated with miRNA mimics or siRNA duplexes. After incubation for 24, 48, 72 and 96 hours, the culture medium was replaced with 100 μL of fresh DMEM. Twenty-five microliters of MTT stock solution (5 g L -1 in phosphate-buffered saline) were added to each well to achieve a final concentration of 1 g L -1 . The plates were incubated for another 4 hours, the culture medium was replaced with dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA), and the absorbance was measured at 570 nm by a SpectraMax M5 Microplate Reader (Molecular Deviced, Sunnyvale, CA, USA). The cell viability was normalized to that of cells cultured in the culture medium without miRNA mimics or siRNA duplexes. Three independent experiments (3 replicates in each) were performed.

Wound healing assay
To determine cell migration, MDA-MB-231 and T47D breast cancer cells transfected with miRNA mimics were seeded in six-well plates, incubated in their respective complete culture medium and grown to confluence overnight. Wounds were made by scraping with a sterilized 200 μL pipette tip, and the debris was rinsed with phosphate-buffered saline. Serial photographs were obtained at 0, 24 and 48 hours using a phase contrast microscope (Olympus IX81, Tokyo, Japan).
MiRNA-transfected cells were scratched using a standard 200 μL tip. The debris was removed by washing with serum-free medium. Serial photographs were obtained at different time points using a phase contrast microscope (Olympus IX81, Tokyo, Japan). Three independent experiments were carried out.

Transwell invasion assays
To determine cell invasion in vitro, Matrigel-coated invasion chambers (8 μm; BD Biosciences, CA, USA) were used according to the manufacturer's protocol. Briefly, miRNA mimic-or siRNA duplex-transfected cells were harvested, re-suspended (1 × 10 5 cells per well) in 200 μL serum-free medium, and transferred to the upper chamber of the Matrigel-coated inserts; culture medium containing 10% FBS was placed in the bottom chamber. The cells were incubated for 24 hours at 37°C; the cells on the upper surface were then removed by peeling off the matrigel and swiping the top of the membrane with cotton swabs. The cells that had invaded the lower surface were fixed and stained with 0.5% crystal violet (Sigma, St Louis, MO, USA) for 30 min, counted under an inverted microscope (Olympus IX71, Tokyo, Japan), and the relative number of invading cells was calculated from five-field digital images taken randomly at 200× magnification. The data are the means ± SD of three independent experiments.

Cell cycle assays
To determine cell cycle distribution, the cells were plated in 6-well plates and transfected with miRNA mimics or siRNA duplexes. After transfection, the cells were collected by trypsinization, fixed in 70% ethanol, washed in PBS, re-suspended in 200 ml of PBS containing 1 mg/ml RNase, 0.05% Triton X-100 and 50 mg/ml propidium iodide (Sigma, St Louis, MO, USA), incubated for 30 min at 37°C in the dark, and analyzed immediately using a FACS-Calibur instrument (Becton Dickinson, CA, USA). The data were analyzed using the CellQuest Pro software (BD Biosciences).

Colony formation assays
After transfecting with miRNA mimics or siRNA duplexes, the cells were seeded in 6-well plates at 5 × 10 2 per well and incubated for 2 weeks for the colony formation assay.
The cells were then washed twice with PBS, fixed with methanol/acetic acid (3:1, v/v), and stained with 0.5% crystal violet (Sigma, St Louis, MO, USA). The number of colonies was counted under the microscope (Olympus IX81, Tokyo, Japan).

Plasmid
The 3′-untranslated regions (3′-UTR) sequences of human FLOT1 containing the putative miR-124 binding sites were isolated from MDA-MB-231 cDNA using PCR amplification and cloned into the pGL3 vector (Promega, Madison, WI, USA), which was termed as wild-type 3′-UTR (wt 3′-UTR). The point mutations in the putative miR-124 binding seed regions were performed using the Quick-Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol. The resultant product served as the mutated 3′-UTR (mut 3′-UTR). Both the wild-type and mutant insert fragments sequences were confirmed by DNA sequencing.
For FLOT1 overexpression, the cDNA of FLOT1 containing the putative miR-124 binding sites was cloned into the multiple cloning site of the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA), which was termed as wild-type 3′-UTR-FLOT1 (wt 3′-UTR-FLOT1). The mut 3′-UTR-FLOT1 was obtained as described above. In the rescue experiment, cells were cotransfected with 50 nM of miRNA mimics and 500 ng of plasmid in a six-well plate.

Luciferase assays
The cells were seeded in triplicate in 24-well plates one day before transfection for the luciferase assays. Wt or mut 3′-UTR vectors and the control vector pRL-TK (Promega, Madison, WI, USA) coding for Renilla lucifearse were co-transfected with miR-124 mimics or negative control into MDA-MB-231 cells using Lipofectamine 2000 reagent, as described previously. After 48 hours of transfection, the cells were harvested and lysed, and the luciferase activity was assayed using the Dual-Glo luciferase assays kit (Promega, Madison, WI, USA). The firefly luciferase values were normalized to Renilla, and the relative ratios of firefly to Renilla activity were reported. Three independent experiments were performed, and the data are presented as the mean ± SD.

Statistical analysis
All experiments were performed at least three times, and all samples were tested in triplicate. The data are shown as the mean ± SEM unless otherwise noted; Student's t-test was used for statistical analysis when only two groups were tested. A one-way analysis of variance was used to compare multiple groups. The difference in miR-124 and FLOT1 expressions between breast cancer specimens and normal adjacent tissues of human subjects was calculated by a two-tailed independent samples t-test. Spearman's correlation analysis was used to determine the correlation between miR-124 and FLOT1 expressions. In all cases, a P < 0.05 was considered statistically significant.