Paired adjacent normal and PTC tumor tissue samples were obtained from 30 PTC patients who underwent surgical resection at the Department of Thyroid Surgery, First Hospital of Jilin University (Changchun, China). All samples were immediately frozen in liquid nitrogen and stored at −80°C until use. The characteristics of the patients are described in Table I. All patients provided written informed consent for the use of their tissues. The present study was approved by the Ethics Committee of Jilin University.

The human PTC cell line, K1, was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and was cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and 1% streptomycin-penicillin (Sigma, St. Louis, MO, USA) at 37°C in a humidified atmosphere containing 5% CO₂.

Total RNAs including miRNAs from tissues and cells were isolated using the miRNeasy Mini kit (Qiagen, Dusseldorf, Germany) according to the manufacturer's protocol. For detection of miR-137, first-strand cDNA was synthesized using miScript reverse transcription kit (Qiagen). The specific primers of miR-137 and U6 were used as previously described (19). U6 small nuclear RNA was used as an internal control. Amplification procedures were performed on an ABI 7900 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) using TaqMan miRNA assay kits (Applied Biosystems, Foster City, CA, USA). To quantify CXCL12, total RNA was reversely transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Dalian, China). Amplification procedures were performed on an ABI 7900 real-time PCR system using real-time PCR mixture reagent (Takara). The specific primers of CXCL12 and GAPDH were used as previously described (20). GAPDH was used as an internal control. The relative gene expression was analyzed using the 2^−ΔΔCt method.

miR-137 mimics (miR-137) and the corresponding miRNA negative control (miR-NC) were purchased from Qiagen (Frederick, MD, USA). The small interfering RNAs (siRNAs) targeting human CXCL12 (si-CXCL12) and the corresponding negative control (si-NC) and the overexpression CXCL12 plasmid were purchased from GenePharma (Shanghai, China). For transfection, K1 cells were seeded into 6-well plates for 24 h and then transiently transfected with these molecular products using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Transfected cells (5×10³ cells/well) were seeded into 96-well plates and cultured in Dulbecco's modified Eagle's medium (DMEM) including 10% FBS. At the indicated times (24, 48 and 72 h), MTT solution (5 mg/ml; Sigma) was added to each well with fresh medium for 4 h. Afterwards, dimethyl sulfoxide (DMSO) (150 µl/well) was added to dissolve the formazan product for ~15 min. The absorption at 570 nm was measured under a multi-well spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA).

Transfected cells were seeded into a 6-well plate in RPMI-1640 medium, supplemented with 10% FBS and 0.3% noble agar at 200 cells/well to form natural colonies. After two weeks, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min, and stained with Giemsa (Sigma). The total number of colonies was counted under a light microscope (Olympus, Tokyo, Japan).

Approximately 1×10⁵ transfected cells were seeded into each well of a 6-well plate. When cell confluence reached ~90–100%, wounds were created in the confluent cells using a 200-µl pipette tip. After wounding, the debris was removed by washing the cells with medium, and then medium was added and cultured for 24 h. Wound healing was observed and photographed at different time points (0, 24 and 48 h) within the scrape line using a phase contrast microscope (Olympus). The results were quantified using ImageJ software.

A Transwell invasion assay was performed with 8.0-mm pore filters according to the manufacturer's instructions (BD Biosciences, San Jose, CA, USA). Briefly, the 2×10⁴ transfected cells were added to the upper Transwell chambers coated with Matrigel (BD Biosciences, Bedford, MA, USA) in serum-free medium. RPMI-1640 medium containing 10% FBS was added into the lower chamber as the chemoattractant. Afterwards, the cells were incubated for 48 h at 37°C in a 5% CO₂ humidified atmosphere. In the present study, the cells that had not migrated through the pores were manually removed from the upper face of the filters using cotton swabs. The cells that had invaded through the membrane were fixed in 90% alcohol and stained with 0.1% crystal violet for 5 min, and then photographed under a microscope at a magnification of ×200 (both from Olympus). The number of invaded cells was counted in five randomly selected fields.

In brief, the miR-137-binding site in the CXCL12 3′-UTR region (wild- or mutant-type) was cloned and inserted into downstream of the firefly luciferase gene in a pGL3-promoter vector (Ambion, Austin, TX, USA). For the luciferase assays, 1×10⁵ K1 cells were seeded into 24-well plates and cultured for 24 h. Then, the cells were co-transfected with 100 ng of wild-type/mutant-type reporter plasmid, and 100 nM of miR-137/miR-NC using Lipofectamine 2000 according to the manufacturer's protocol. At 48 h after transfection, Renilla luciferase activities in the cell lysates were measured using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA).

The transfected cells were harvested, washed and lysed with RIPA buffer (Beyotime, Shanghai, China). Protein concentrations were measured using the bicinchoninic acid protein assay kit (Beyotime). Equivalent quantities (30 µg) of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline for 2 h and incubated overnight at 4°C with primary antibodies against CXCL12 (1:1,000) and GAPDH (1:5,000) (both from Santa Cruz). The membranes were washed 3 times in TBS-Tween-20 and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at a 1:5,000 dilution for 1 h. The membranes were washed and probed with the secondary antibody conjugated to horseradish peroxidase (Santa Cruz) at a 1:5,000 dilution for 2 h at room temperature. Proteins were visualized using chemiluminescence detection (SignaGen, Rockville, MD, USA).

The data are expressed as the mean ± SD (standard deviation) of 3 independent experiments. Group differences were compared using two-tailed Student's t-test or one-way ANOVA from SPSS version 19.0 software (SPSS, Inc., Chicago, IL, USA). A P-value of <0.05 was considered to indicate a statistically significant result.

To determine whether miR-137 is involved in PTC tumorigenesis, we detected the expression levels of miR-137 in the PTC and corresponding adjacent normal tissues. qRT-PCR assay showed that expression of miR-137 was significantly decreased in the PTC tissues compared with that in the adjacent normal tissues (Fig. 1). To further investigate the clinical relevance of miR-137 in PTC, the median value (0.46) of all 30 PTC samples was chosen as the cut-off point for grouping PTC patients with low miR-137 expression (<0.46, 16 cases) and high miR-137 expression (>0.46, 14 cases). Then, by the Chi-square test, correlations between miR-137 expression and clinicopathological parameters were analyzed. It was found that miR-137 expression was significantly negatively associated with tumor-node-metastasis (TNM) stage (P<0.01) and lymph node metastasis (P<0.01), which are both indicators of poor prognosis (Table I). However, no significant correlations were found between miR-137 expression and patient age and gender, as well as tumor size.

To investigate the biological function of miR-137 in PTC, we restored its expression by transfection of the miR-137 mimic in the K1 cells (Fig. 2A). Then, cell proliferation and colony formation were determined in the K1 cells after transfection with miR-137 or miR-NC. It was found that restoration of miR-137 in K1 cells significantly reduced cell proliferation (Fig. 2B) and colony formation (Fig. 2C)

To investigate whether miR-137 has an effect on migration and invasion in K1 cells, wound heal and Transwell chamber assays, respectively, were performed in K1 cells after transfection with miR-137 or miR-NC. It was found that overexpression of miR-137 in the K1 cells significantly suppressed cell migratory (Fig. 3A) and invasive (Fig. 3B) capabilities.

We investigated the candidate targets for miR-137 using prediction algorithms (Targetscan6.2 and miRanda). We selected CXCL12 for further validation since CXCL12 mRNA has one potential complimentary miR-137 binding site within its 3′-UTR region (Fig. 4A). We then performed a luciferase-based assay to validate whether this gene was regulated by miR-137. It was found that restoration of miR-137 expression in K1 cells obviously suppressed the luciferase activity of the wild-type CXCL12 site, while activity of the mutant CXCL12 site was not affected (Fig. 4B), which suggested that CXCL12 is directly targeted by miR-137. To determine whether miR-137 regulates CXCL12 expression in PTC cells, CXCL12 expression at the mRNA and protein levels was determined in K1 cells transfected with miR-137 or miR-NC by qRT-PCR and western blotting, respectively. As expected, restoration of miR-137 in K1 cells obviously inhibited CXCL12 expression at the mRNA level (Fig. 4C) and protein level (Fig. 4D). These results indicated that CXCL12 is a direct target of miR-137 in PTC cells.

The above results suggested that CXCL12 is a direct target of miR-137 in PTC cells. Thus, we investigated the expression of CXCL12 in PTC and corresponding normal tissues by qRT-PCR and western blotting. CXCL12 expression was upregulated at the mRNA level (Fig. 5A) and protein level (Fig. 5B) in the PTC tissues compared with levels in the adjacent normal tissues. In addition, the miR-137 mRNA expression level was inversely correlated with miR-137 expression in the PTC tissues by Spearman's correlation analysis (Fig. 5C; r=−0.549; P<0.01).

To explore the biological functions of CXCL12 in PTC cells, endogenous expression of CXCL12 was knocked down in K1 cells with specific siRNA against CXCL12 (si-CXCL12), and then cell proliferation, colony formation, migration and invasion were assessed. Knockdown of CXCL12 significantly inhibited cell proliferation (Fig. 6A), colony formation (Fig. 6B), as well as decreased cell migration (Fig. 6C) and invasion (Fig. 6D) capabilities, suggesting that inhibition of CXCL12 mimicked the inhibitory effect of miR-137 overexpression in the K1 cells.

To evaluate whether the negative role of miR-137 on PTC was due to its regulation of CXCL12, we overexpressed miR-137, which subsequently strengthened CXCL12 expression in K1 cells. Our results showed that forced CXCL12 expression partially abrogated the inhibitory effects of miR-137 on cell proliferation, colony formation, migration and invasion (Fig. 7A–D). These results indicated that miR-137 exerts suppressive effects on PTC cells partially by targeting CXCL12.