The hsa-miR-100 (miR-100) precursor was synthesized by Songon Tech (Beijing, China) and subcloned into the pcDNA6.2-GW/EmGFP-miR vector (referred to as pcDNA6.2/miR-100) according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Scramble miRNA was cloned into the same vector and used as a negative control (named pcDNA6.2/miR-NC). The sequence of the control miRNA was: 5′-AGGTACGAAACGCTAAGAAT-3′. To construct the luciferase reporter vector, the human CXCR7 3′-UTR containing putative binding sites for miR-100 was amplified by PCR and cloned in pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA). The construct was named pmiR-GLO-CXCR7. The primers were as follows: forward primer, 5′-GGGAGCTCTCTGCCCTGGAGAGGCTCTG-3′ and reverse primer, 5′-CGTCTAGACAAAACTGAAGTCACGCTA-3′. The accuracy of all cloning was confirmed by sequencing.

Human esophageal squamous cancer cell line (Ec-109) (kindly provided by Dr Ling Zhou from the Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention) was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified incubator with 5% CO₂ at 37°C. All cell culture materials were obtained from Gibco, USA. Lipofectamine 2000 (Invitrogen) was used for cell transfection. Briefly, cells at ~60% confluency in 6-well plates were transfected with the plasmid pcDNA6.2/miR-100 or control vector pcDNA6.2/miR-NC. Blasticidin (4 µg/ml) was applied to select stable miR-100-overexpressing clones. The expression of miR-100 was confirmed by real-time PCR after 3 weeks of selection from individual clones.

The expression of mature miR-100 and the primary transcript of CXCR7 was analyzed by RT-qPCR. Total RNA was extracted from cultured cells by TRIzol reagent (Invitrogen) based on the provided protocols. Total RNA (1 µg) was applied for reverse transcription to synthesize miRNA cDNA using NCode™ VILO™ miRNA cDNA synthesis kit (Invitrogen). RT-qPCR analysis was performed using EXPRESS SYBR^®-GreenER™ miRNA RT-qPCR kit (Invitrogen) based on instructions. Reverse transcription mix (20 µl) was amplified by PCR with denaturation at 95°C for 2 min and 40 cycles at 95°C for 10 sec and 60°C for 1 min. U6 was applied as an endogenous control for normalization. Each sample was analyzed in triplicate. The forward primers that target miR-100 or U6 were purchased from Songon Tech.

To analyze the expression of CXCR7, 1 µg of total RNA was reverse transcribed in a final volume of 20 µl to synthesize first-strand cDNA using ImProm-II™ reverse transcription system (Promega) based on the instructions of the manufacturer. The transcript levels were detected using Brilliant^® II SYBR^®-Green qPCR Master Mix (Stratagene, USA) to monitor amplification. β-actin served as an endogenous control for normalization. PCR reactions (25 µl) in triplicate were carried out by an initial denaturation at 95°C for 10 min followed by 40 cycles, each consisting of 30 sec at 95°C, 30 sec at 58°C, 30 sec at 72°C, and then 1 cycle for melting curve consisting of 1 min at 95°C, 30 sec at 55°C, 30 sec at 95°C. Primer sequences used were as follows: 5′-GGCTATGACACGCACTGCTACA-3′ (forward primer for CXCR7) and 5′-TGGTTGTGCTGCACGAGACT-3′ (reverse primer for CXCR7); 5′-AGAAAATCTGG CACCACACC-3′ (forward primer for β-actin) and 5′-TAGCACAGCCTGGATAGCAA-3′ (reverse primer for β-actin). The 2^−ΔΔCt method for relative quantification of gene expression was used to determine miRNA and CXCR7 mRNA expression levels. RT-qPCR primers were designed using Primer Express software (version 5.0; Applied Biosystems).

Cells (2,000 cells/well) were passaged in 96-well plates in RPMI-1640 culture medium containing 10 µg/µl blasticidin, supplemented with 10 or 7% FBS. Cell proliferation were measured by the OD values using Cell Counting Kit-8 (CCK-8) (Dojindo, Japan). The absorbance was measured at 450 nm on a microplate reader (PerkinElmer, USA), and the reference light was 650 nm. Every sample was measured for three times.

Transwell insert chambers (Corning, Corning, NY, USA) with 8-µm pores were used to conduct these assays. For the migration assay, 1×10⁵ cells were seeded into the upper chamber in serum-free medium in triplicate. Medium containing 20% FBS in the lower chamber served as the chemoattractant. After incubation for 24 h at 37°C in a 5% CO₂ humidified incubator, cells in the upper chambers were removed by wiping with a cotton swab and cells that had migrated to the lower surface of the filter were fixed with 4% formaldehyde overnight at 4°C and stained with 0.2% crystal violet for 10 min. Cell migration was scored by counting five random fields/filter under a light microscope. For the invasion assay, 3×10⁵ cells were plated into upper chambers precoated with Matrigel (BD, USA) in serum-free medium in triplicate. Medium with 20% FBS was added to the lower chamber to serve as the chemoattractant. After incubation for 24 h at 37°C, non-invading cells on the upper surface of the filter were removed by wiping with a cotton swab and invading cells that had migrated to the lower surface of the filter were fixed, stained and scored as described above.

Cells (2,000) were seeded in 1.5 ml of standard growth medium containing 0.33% low-melting-temperature soft agar (SeaKem; FMC Corporation) and plated onto a 3 ml layer of solidified 0.66% soft agar in the same medium in a 35-mm tissue culture dish. Cultures were fed once a week with 0.5 ml of complete medium. Each cell clone tested was seeded in triplicate. The cell colonies becoming visible microscopically after 3±4 weeks were scored weekly, and photographed with a Olympus microscope (Japan).

Female athymic BALB/c nude mice ~4–6 weeks old were purchased from the Laboratory Animal Center of the Army Research Center (Beijing, China), and carefully fostered under the guidelines for the care and use of laboratory animals of the local government. All mice were housed under a pathogen-free condition. All experiments were performed with guidelines provided by the National Cancer Insitute (NIH; USA). For xenografts, 10 mice were randomly separated into 2 groups. Cells (2×10⁶) that stably expressed miR-100 or miR-NC were suspended in serum-free RPMI-1640 medium and injected subcutaneously into the flank, respectively. Five weeks post-injection, the mice were sacrificed and photographed. Tumor volumes (cm³) were calculated using the following standard formula: [length × width] × [(length + width)/2].

Two groups of Ec-109 cells (stably overexpressing miR-100 or miR-NC) were seeded in 6-well plates for ~24 h to reach 60% confluency, and then transiently transfected with the pmiR-GLO-CXCR7 gene report vector for 12 h. Empty vector (pmiR-GLO-NC) was used as a control. Luciferase activity was analyzed using the Dual-Glo^® Luciferase Assay system (Promega), which was analyzed by three independent experiments performed in triplicate.

Proteins were extracted from the miR-100-expressing or the control cells using the Membrane and Cytosol Protein Extraction kit (Beyotime, China). Protein concentrations were determined by the BCA protein assay kit (Tiangen, China). Protein samples were denatured by boiling for 5 min and were loaded onto SDS-PAGE (10% polyacrylamide gel) for electrophoresis, and then transferred onto methanol-activated polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, USA). Non-specific reactivity on the membranes was blocked by 5% skim milk [non-fat dry milk in Tris-buffered saline with Tween-20 (TBST)] overnight at 4°C, and then washed with TBST. Primary antibodies for human β-actin (Cell Signaling Technology, Danvers, MA, USA) or CXCR7 (Bioss, China) were incubated with the membranes overnight at 4°C, respectively. The membranes were then incubated with the secondary antibody, conjugated with fluorescent dyes: IRDye 800CW (KPL, Gaithersburg, MD, USA) for 1 h. After washing, the membranes were developed under the Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, NE, USA) to obtain the blotting images.

Data are presented as mean ± SD of at least three independent experiments. The differences between group were analyzed using the Student's t-test. A p-value of <0.05 was considered statistically significant for all tests. Analyses were performed using statistical analysis software SPSS 19.

In the present study, the miR-100 precursor and scramble miRNAs were exogenously expressed in esophageal cancer cells and stable miRNA-expressing cells were acquired by blasticidin. The ectopic expression of mature miR-100 in the transfectants was initially confirmed by qRT-PCR (Fig. 1A). To investigate the potential regulatory effects of miR-100 on esophageal cancer cells, CCK-8 and Transwell assays were used to evaluate the proliferation, migration and invasion of the esophageal cancer cells upon miR-100 transfection, respectively. The data showed that miR-100 overexpression significantly inhibited cell growth when cells were cultured in 7% of FBS compared to the scramble miRNA (control) (Fig. 1B). However, no obvious inhibition in cell growth was observed when cultured in 10% of FBS (Fig. 1C). Moreover, compared with the control, ectopic expression of miR-100 was able to suppress the migration (Fig. 2A and B) and invasion (Fig. 2C and D) of the esophageal cancer cells, implying a tumor-suppressor role of miR-100 in esophageal cancer.

An anchorage-independent growth assay and xenograft transplantation experiment were further conducted to examine the effects of miR-100 on tumorigenesis in esophageal cancer cells. The results showed that large cell colonies were generated in the scramble miRNA-transfected cells (Fig. 3A). In contrast, overexpression of miR-100 inhibited colony formation and significantly reduced colony numbers in vitro when compared to the control (Fig. 3B and C). In vivo xenograft transplantation analysis further showed that miR-100 overexpression markedly suppressed tumor growth in nude mice (Fig. 4A and B). The tumor volume was much smaller than that of the controls (Fig. 4C), which was consistent with the in vitro colony formation assay. The obtained data suggest that miR-100 plays a suppressive role in the tumorigenesis of esophageal cancer.

Several computational prediction websites such as TargetScan, PicTar and MiRanda and the target prediction methods as previously reported (12,13) were used to identify functionally relevant targets of miR-100. The expression of CXCR7 which contains a putative target sequence of miR-100 in 3′-UTR was quantitatively examined by both RT-PCR and western blotting. The data showed that the expression of CXCR7 was significantly decreased in the miR-100-expressing cells compared to that noted in the control cells (Fig. 5A and B). Luciferase assay was thereafter used to investigate whether CXCR7 is a direct target of miR-100. The 3′-UTR region of the CXCR7-encoding gene containing the putative binding site of miR-100 was subcloned into the luciferase reporter vector and co-transfected with miR-100 into esophageal cancer cells. Cells co-transfected with the empty vector and miR-100 were used as a control. The results showed that luciferase activity was markedly reduced in the reporter construct containing 3′-UTR of CXCR7 compared to that in the empty vector control (Fig. 5C), indicating that CXCR7 is a direct downstream target gene of miR-100 and miR-100 downregulates the expression of CXCR7 through direct binding to its 3′-UTR (Fig. 5D).