From October 2013 to March 2014 a total of 22 subjects recruited from October 2013 to March 2014 took part in the present study. A total of 12 patients diagnosed with hyperthyroidism were recruited from the Department of Endocrinology and 10 healthy subjects were recruited from the Physical Examination Center at The 82nd Hospital of Chinese People's Liberation Army (CPLA). Patients who had diabetes mellitus or other endocrine diseases, or with a body mass index >30 kg/m², were excluded from taking part in the study. Patient details are shown in Table I. Patients taking any drugs known to influence TH metabolism were additionally excluded. The ethics committee of The 82nd Hospital of CPLA approved the present study. Written informed consent was obtained from all subjects.

The clinical characteristics of the study population include in serum free triiodothyronine (FT3), serum free thyroxine (FT4), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride content (TG) and total cholesterol (TC). These were measured by Olympus AU640 automatic biochemical analyzer (Olympus Corporation, Tokyo, Japan).

Human hepatoblastoma (HepG2) cell line is known to be misidentified, originally thought to be a hepatocellular carcinoma cell line but shown to be from an hepatoblastoma (10). HepG2 is a perpetual cell line, which was derived from the liver tissue of a 15-year-old Caucasian American male with a well-differentiated hepatocellular carcinoma. HepG2 cells are a suitable in vitro model system for the study of polarized human hepatocytes. HepG2 cells and their derivatives are also used as a model system for studies of liver metabolism and toxicity of xenobiotics. HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin. For treatment with T3 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), cells were seeded in 12-well culture plates at a density of 3×10⁵ cells/well, and maintained for 2 days incubated at 37°C in a humidified chamber containing 5% CO₂. Following overnight incubation in serum-free DMEM, cells were treated with 100 nM T3 for 12, 24 or 36 h. For the investigation of triglyceride synthesis, the medium was supplied with a 0.25 mM free fatty acid (FFA) cocktail composed of lauric acid, myristic acid, linoleic acid, oleic acid and arachidonic acids (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).

HepG2 cells, grown to 70–80% confluence, were transiently transfected with miR-206 mimic (sense, 5′-UGGAAUGUAAGGAAGUGUGUGG-3′ and antisense, 5′-ACACACUUCCUUACAUUCCAUU-3′) or miR-206 inhibitor (5′-CCACACACUUCCUUACAUUCCA-3′), or a corresponding mimic negative control inhibitor (5′-UUCUCCGAACGUGUCACGUTT-3′; Shanghai GenePharma Co., Ltd., Shanghai, China), using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. All the mimic and inhibitor containing FAM reporter fluorophores were transfected at 10 nM. Transfection efficiency was observed using an inverted fluorescent microscope (original magnification, ×400; Leica Microsystems GmbH, Wetzlar, Germany), and evaluated using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The mixtures were added to the culture, and the culture medium was changed after 8 h, and cultured in DMEM supplemented with 10% fetal bovine serum for 36 h further for subsequent experiments.

Cell/serum total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) or QIAzol reagent (Applied Biosystems; Thermo Fisher Scientific, Inc.), followed by isolation and purification using a miRNeasy Mini kit/miRNeasy serum kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Subsequently, first-strand cDNA was synthesized by RT using a TaqMan miRNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression of miR-206 was analyzed by qPCR using TaqMan Universal Master Mix II in the ABI 7500 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). All primers were provided in the TaqMan^® MicroRNA Assays (cat. no. 4427975; Thermo Fisher Scientific, Inc.). PCR conditions were as follows: 50°C for 2 min, 95°C for 2 min, and then 40 cycles of 94°C for 15 sec and 60°C for 1 min. U6 small nucleolar RNA was used as an internal standard for normalization. miR-206 expression was calculated using the comparative quantification cycle (Cq) method (2^−ΔCq; ΔCq = Cq_miR-206 - Cq_U6), the Cq values were converted into RQ via the 2-ΔΔCq method (11), incorporating the calculated amplification efficiency for each primer pair.

Oil Red O stock solution was prepared in isopropanol (0.25 g/100 ml) and heated to 100°C for 10 min. HepG2 cells grown to 70–80% confluence were washed with PBS and fixed using 4% paraformaldehyde for 30 min at room temperature. Cells were soaked in filtered 0.6% (w/v) Oil Red O solution (60% Oil Red O stock solution, 40% water) for 20 min at room temperature. Cells were washed with water to remove unbound dye and visualized using light microscopy (original magnification, ×400).

Each sample was analyzed in triplicate. All data are presented as the mean ± standard error of the mean. Student's t-test was used to analyze the differences between groups. The correlation between T4 parameters and miR-206 expression levels was analyzed using the Pearson product-moment correlation coefficient method. Comparisons among multiple groups were statistically performed using one-way analysis of variance followed by Dunnett's test. Two-tailed P<0.05 was considered to indicate a statistically significant difference. The data were analyzed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA).

The clinical characteristics of the study population are presented in Table I. Patients with hyperthyroidism exhibited increased serum free T3 (FT3) and free T4 (FT4) concentrations compared with the control subjects.

The RT-qPCR analysis demonstrated that the serum miR-206 levels in patients with hyperthyroidism were significantly decreased compared with euthyroid control subjects (Fig. 1). The present study further analyzed the correlation between TH and miR-206 expression in patients with hyperthyroidism. No apparent correlation was observed between miR-206 expression and serum FT3 or FT4 levels (data not shown).

In order to clarify the effect of TH on miR-206 expression, HepG2 cells were treated with 100 nM T3 for 12, 24 or 36 h, with miR-206 expression examined using RT-qPCR analysis. The expression of miR-206 was significantly decreased 24 h subsequent to the treatment with T3, compared with HepG2 cells without T3 (Fig. 2). The expression of miR-206 remained reduced at 36 h following the addition of T3 (Fig. 2).

To validate the effects of T3 on lipid synthesis, HepG2 cells were cultured in DMEM supplemented with a 0.25 mM FFA cocktail and treated with 100 nM T3 for 12, 24 or 36 h. Oil Red O staining demonstrated that T3 suppressed lipid accumulation in HepG2 cells (Fig. 3A). The TG was significantly reduced 12 h subsequent to treatment with T3 (Fig. 3B), while the TC content was significantly reduced 24 h subsequent to treatment with T3 (Fig. 3C). The TG and TC content of the HepG2 cells remained reduced 36 h following treatment with T3.

The transfection of HepG2 cells with miR-206 inhibitor confirmed that the inhibitor effectively knocked down miR-206 expression (Fig. 4A). The addition of the miR-206 inhibitor reduced the TG at 24 h, and TG and TC content at 36 h in HepG2 cells (Fig. 4B).

The transfection of HepG2 cells with the miR-206 mimic or mimic negative control confirmed that the miR-206 mimic increased miR-206 expression (Fig. 5A). HepG2 cells treated with T3 exhibited a reduced TG content that was restored with miR-206 overexpression (Fig. 5B). The addition of mimic negative control did not alter TG in T3-treated cells.