Human visceral pre-adipocytes (ScienCell Research Laboratories, San Diego, CA, USA) were maintained in preadipocyte medium containing 5% fetal bovine serum, 1% preadipocyte growth supplement, and 1% penicillin/streptomycin solution (PAM, cat. no. 7211; all ScienCell Research Laboratories) at 37°C in an incubator under 5% CO₂ and 95–100% humidity (16). To induce differentiation, serum-free PAM (containing 50 nM insulin, 100 nM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 100 mM rosiglitazone) was added to confluent human preadipocytes (day 0), and the medium was replaced every 2 days for the next 4 days. Thereafter, the medium was replaced with serum-free PAM containing 50 nM insulin and replaced every 2 days until lipid droplets had accumulated in cells (day 15) (17). Differentiated adipocytes were used for experiments 15 days later, when >80% of cells demonstrated morphological and biochemical properties of adipocytes. After an overnight incubation in serum-free PAM, human adipocytes were treated with a final concentration of 10 ng/ml TNF-α (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or 30 ng/ml IL-6 (Sigma-Aldrich; Merck KGaA, Germany) for 0, 4, 8 or 24 h at 37°^C in an incubator under 5% CO₂ and 95–100% humidity (18). Human embryonic kidney 293T (HEK293T) cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Bovogen Biologicals, Keilor East, Victoria, Australia). HEK293T cells were also incubated at 37°C in an incubator under 5% CO₂ and 95–100% humidity (17).

Total miRNA from human adipocytes was extracted using an miRNeasyMiNi kit (Qiagen GmbH, Hilden, Germany). The quality of microRNA was assessed using 1% agarose gel electrophoresis and the concentration of microRNA was measured using spectrophotometry at 260 nm. Equal quantities of microRNA (200 ng) were used to synthesize cDNA using TaqMan microRNA Reverse Transcriptase kit (Applied Biosystems; Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's protocol (Table I). qPCR with SYBR (Power SYBR^™ Green PCR Master Mix; cat. no. 4368577; Thermo Fisher Scientific, Inc.) was carried out using an Applied Biosystems 7500 Sequence Detection system (ABI 7500 SDS; Thermo Fisher Scientific, Inc.) following manufacturer's protocol (Table II). Relative gene expression was quantified using the 2^−ΔΔCq method (18). Total microRNA expression was normalized to small nuclear (sn)RNA U6. Primer cat. numbers are: 001973 for snRU6 and 002840 for miR-1275 (Applied Biosystems; Thermo Fisher Scientific Inc.).

Hsa-miR-1275 precursor (pri-miR-1275) sequence was obtained from Ensembl (http://www.asia.ensemble.org/index.html). The precursor sequence was numbered from +1 to +80, and therefore 1,500 bp upstream of pri-miR-1275 was considered to be the predicted promoter region. To identify the predicted binding sites of NF-κB, the sequence of the predicted promoter was uploaded to Genomatrix (http://www.genomatix.de), Jaspar (jaspar.genereg.net), and Promo_v3 (alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3), and the intersection of putative binding sites was recorded (19–21).

The predicted promoter region of miR-1275 was amplified by PCR with TaqDNA polymerase (cat. no. DR100A; Takara Biotechnology Co., Ltd., Dalian, China). DNA was amplified at 95°C for 40 sec for denaturation, 55°C for 35 sec for renaturation and 130 sec for elongation with 30 cycles. The site-specific mutation vectors aimed at the binding site of NF-κB were constructed by introducing point mutations with overlap-extension PCR. The predicted binding sites of NF-κB are presented in Table III. PCR products were then cloned into the dual-luciferase reporter plasmid PEZX-FR01 using MluI/BamHI restriction sites (GeneCopoeia Inc., Rockville, MD, USA). The primer sequences used are listed in Table IV.

HEK293T cells were cultured in 24-well plates at density of 2×10⁴ cells/well. When grown to a density of 60–70%, they were transfected with 2 µg/well reporter plasmids and 6-µl/well Lipofectamine (Invitrogen; Thermo Fisher Scientific Inc.) for 6 h. Empty PEZX-FR01 plasmid served as a control. Following this, cells were washed with serum-free medium and treated with the NF-κB activator TNF-α (10 ng/ml) for 24 h. For the rescue assay, HEK293T cells were treated with the NF-κB inhibitor 4-methyl-N1-(3-phenyl-propyl) -benzene-1,2-diamine (10 µM; Calbiochem; Merck KGaA) for 1 h. Subsequently, cells were washed with serum-free medium and treated with TNF-α (10 ng/ml) for 24 h. After this, luciferase activity was analyzed using a dual luciferase reporter assay (Promega Corporation, Madison, WI, USA) with a GloMax 96 Microplate Luminometer E6501 (Promega Corporation) (22). Firefly luciferase activity was normalized to the Renilla luciferase activity.

The data were analyzed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). All data are presented as the mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance followed by Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Human mature adipocytes were treated with 10 ng/ml TNF-α or 30 ng/ml IL-6 for 24 h, and the expression of miR-1275 was examined at different times (0, 4, 8 and 24 h). The expression of miR-1275 was normalized to the expression of snRU6. After treatment with TNF-α, miR-1275 relative expression was significantly lower than in untreated control cells (Fig. 1A). A similar reduction in miR-1275 expression was also observed in human adipocytes treated with IL-6 (Fig. 1B). These results indicated that TNF-α and IL-6 could regulate the expression of miR-1275 in human adipocytes.

Bioinformatic analysis was performed on the 1,500 bp upstream sequence of pri-miR-1275 to further determine how TNF-α and IL-6 regulate miR-1275 expression. Several binding sites of NF-κB, the downstream transcription factor of TNF-α and IL-6, were identified on the 1,500 bp sequence of the predicted promoter. This suggested that TNF-α and IL-6 may regulate miR-1275 expression through NF-κB. To locate the strongest binding site of NF-κB, the sequence of putative promoters was submitted to the following websites: Genomatrix, Jaspar and Promo_v3. Two sites (from −806 to −792 and from-288 to-274) were predicted by all three websites (Table II). Therefore, these were chosen to be the strongest binding sites of NF-κB (Fig. 2).

To determine whether the two predicted binding sites were functional, different promoters were made and their activity was examined in HEK293T cells. For the deletion assay (Fig. 2A), dual-luciferase reporter plasmids PEZX-FR01 of Pro-1 (including both sites), Pro-2 (only including the-288 site) and Pro-3 (including neither site) were transfected into HEK293T cells. Pro-1 demonstrated >10 times higher luciferase activity compared with the empty reporter plasmids, whereas Pro-2 and Pro-3 showed similar luciferase activity compared with the control (Fig. 3A). These results suggested that the sequence from −840 to +1 was the promoter region of miR-1275, and the −806 site was necessary for the promoter's activity (Fig. 3B and C).

To confirm the role of −288 site, the core binding sequences of the −806 and −288 sites were mutated (Fig. 4A and B). The luciferase activities of Mut-1 (mutation on-806 site), Mut-2 (mutation on-288 site) and Mut-3 (mutation on both-806 and-288 sites) demonstrated no difference compared with the empty reporter plasmids (Fig. 4C). These results suggested that the −288 site was also necessary for the promoter activity.

To confirm the above-mentioned results, a rescue assay was performed. HEK293T cells were transfected by Pro-1-PEZX-FR01 and then treated with JSH-23 before treatment with TNF-α. As presented in Fig. 5, cells treated with JSH-23 and TNF-α demonstrated significantly more luciferase activity than cells treated with TNF-α alone. These results confirmed that TNF-α decreased miR-1275 promoter activity through NF-κB.