Patients and sample collection
The protocols of this study were approved by the Ethics Committee of Peking University First Hospital University. This study enrolled 120 obese patients, including 60 cases with IR and 60 cases without IR (simple obesity), from the hospital between 2016 and 2017. In addition, 40 healthy individuals were recruited, who underwent routine health examination in the hospital. None of the healthy controls had IR or any other metabolic diseases. All the participants provided the written informed consents. Venous blood samples were collected from the patients and healthy controls, and serum specimens were extracted from the blood using centrifugation. The fasting blood glucose (FBG) and fasting serum insulin (FINS) were measured and used for the calculation of homeostasis model of assessment for IR index (HOMA-IR).
Cell culture and transfection
Mouse 3 T3-L1 pre-adipocytes were purchased from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS). For the differentiation of pre-adipocytes, the cells were incubated in induction medium I (10 μg/mL of insulin, 0.5 mM 1-methyl-3-isobutyl-xanthine and 1 μg/mL of dexamethasone in DMEM supplemented with 10% FBS) for 24 h after they spread to all the bottom of the culture plates. After a further 24 h incubation in fresh DMEM supplemented with 10% FBS, the cells were cultured with the induction medium II (10 μg/mL of insulin in DMEM with 10% FBS0 for 48 h. Lastly, the cells were cultured in fresh DMEM with 10% FBS, which was renewed 2 days once until the mature adipocytes accounting for 95% of the cells.
To regulate the expression of miR-214 in 3 T3-L1 cells, miR-214 mimic (ACAGCAGGCACAGACAGGCAGU), miR-214 inhibitor (ACUGCCUGUCUGUGCCUGCUGU) or miRNA negative control (miR-NC, UUCUCCGAACGUGUCACGU) (GenePharma, Shanghai, China) was transfected into the cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The cells transfected with only transfection reagent were set as the mock group.
RNA extraction and quantitative real-time-PCR (qRT-PCR)
Total RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Single stranded cDNA was synthesized from the RNA using a PrimeScript RT reagent kit (Takara, Shiga, Japan). The expression of miR-214 was measured using qPCR, which was carried out using a SYBR green I Master Mix kit (Invitrogen, Carlsbad, CA, USA) and the ABI Prism 7300 system (Thermo Fisher Scientific, Waltham, MA, USA). Following are the sequences of primer: miR-214 F: 5′-GCCGAGACAGCAGGCACAG-3′, R: 5′-CTCAACTGGTGTCGTGGA-3′; U6 F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-AACGCTTCACGAATTTGCGT-3′. The relative expression values were calculated by 2−ΔΔCt method and normalized to U6.
Luciferase reporter assay
DPP4 was predicted as a target gene of miR-214 using TargetScan (http://www.targetscan.org/vert_72/). The 3′-UTR of DPP4 was combined into the psiCHCK-2 vector as the wild-type vector (DPP4-WT), and the mutant 3′-UTR was used to construct the mutant-type vector (DPP4-MT). The 3 T3-L1 cells were seeded into 24-well plates and co-transfected with miR-214 mimic, miR-214 inhibitor or miR-NC and DPP4-WT or DPP4-MT. Luciferase activity was estimated using a dual-luciferase reporter assay system (Promega, Madison, WI, USA).
Enzyme-linked immune sorbent assay (ELISA)
The concentration of DPP4 was estimated using the Human sCD26 Platinum ELISA kit (eBioscience, Vienna, Austria) as per the manufacturer’s protocols.
Obesity-induced IR animal model and treatment
Total of 36 Sprague-Dawley (SD) rats (males, 6 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, China). After a one-week adaptation, the rats were used for model construction. The protocols for animal experiments were approval by the Animal Ethics Committee of Peking University. The rats were randomly divided into 2 groups, including normal control (NC) group (n = 6) and IR model group (n = 30). The animals in the NC group were fed with regular diet (10% fat). The rats in the IR model group were fed with D12492 high-fat diet (HFD; 60% fat) for obesity-associated IR model construction. The body weight (BW) of the rats was recorded during the 12 weeks of feeding, as well as the FBG and FINS.
The IR model group was further divided into 4 groups: IR control group (n = 6), vildagliptin (VG) group (n = 6), miR-214 mimic group (n = 6) and VG + miR-214 mimic group (n = 6). The animals in the IR control group were given normal saline by gavage. The rats in the VG group were oral injected with 3 mg/kg VG (Novartis, Basel, Switzerland). In the miR-214 mimic group, Lentivirus (100 μL, 2 × 107 TU/mL) with miR-214 mimic were intraperitoneally injected in the rats. In the VG + miR-214 mimic group, the animal received both VG and miR-214 mimic treatments. The blood and omental adipose tissues were collected from the rats and stored at − 80 °C for further examination.
Obesity-associated IR cell model and treatment
After differentiation, the mature 3 T3-L1 adipocytes were grouped into untreated group and palmitic acid (PA) group. The cells in untreated group received no treatment as controls. The cells in PA group received stimulation of 1 mM PA for the construction of IR cell model.
To evaluate the effects of miR-214 on IR development in vitro, the IR model cells were also divided into IR control (IR control-C) group, VG (VG-C) group, miR-214 mimic (miR-214 mimic-C) and VG + miR-214 mimic (VG + miR-214 mimic-C) group. In the VG-C group, IR model cells were treated with 50 nM DPP4 for 24 h. In the miR-214 mimic-C group, the miR-214 mimic was transfected into the adipocytes using Lipofectamine 2000 before 48 h of PA treatment. The cells in the VG + miR-214 mimic-C group were received the treatment with both VG and miR-214 mimic.
Oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT)
OGTT and IPITT were performed to confirm the construction of IR animal model and to evaluate the effects of miR-214 on the development of IR. For OGTT, the animals free accessed to water and were fasted for 12 h, and then treated with 40% of glucose (2 g/kg BW) via oral injection. A glucose meter (ACCU-CHEK Performa Roche, Germany) was used to examine the blood glucose at the tip of the tail at 0, 30, 60, 90, 120 min after the administration of glucose. The area under the curve (AUC) was calculated to evaluate the sensitivity of islet β-cells. For IPITT, the rats free accessed to water and were fasted for 12 h, and then treated with insulin (0.75 IU/kg BW) through intraperitoneal injection. A glucose meter was used to examine the blood glucose at the tip of the tail at 0, 30, 60, 90, 120 min after the administration of glucose. The AUC value was also computed to evaluate the insulin sensitivity.
Glucose uptake assay
To analyze the IR conditions of the adipocytes, glucose uptake assay was performed using a glucose colorimetric/fluorometric assay kit (Biovision, Inc., Milpitas, CA, USA). In briefly, the cells were incubated with or without insulin (100 nM) at 37 °C for 20 min. Then the glucose assay buffer was added into the cells and incubated at 37 °C for 10 min. The absorbance at 570 nm was measured by a microplate reader (BioTek, Beijing, China).
All the data in this study were expressed as mean ± SD and analyzed using SPSS 21.0 software (SPSS Inc., Chicago, IL) and GraphPad Prism 7.0 software (GraphPad Software, Inc., USA). Comparisons between groups were assessed by Student’s t test or one-way ANOVA followed by Turkey’s post hoc test. Pearson correlation analysis was used to analyze the correlation between two parameters. The diagnostic potential of miR-214 was evaluated using a receiver operating characteristic curve (ROC). A difference with a P < 0.05 was considered statistically significant.