Cell culture
HUVECs were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA). HUVECs were grown in a humidified environment at 37°C supplemented with 5% CO2, and prepared for our subsequent experiments. Cells at passages 3-9 were applied in our study.
Morphological observation
33.3 mmol/L of glucose was determined as high-concentration based on previous literatures [19, 20]. To determine the effect of high-glucose on cells, cultured HUVECs were obtained and placed in serum-free medium containing 33.3 mmol/L of high-glucose. For comparison, HUVECs only incubated in serum-free medium were served as controls. After 24 h, the morphology of HUVECs incubated with or without high-glucose was observed under the inverted microscope (Eclipse TS-100, Nikon, Tokyo, Japan).
Cell treatment
To investigate the role of glucose in miR-145 expression, HUVECs were respectively treated with different concentrations of glucose (15, 30, 45 mmol/L). Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to determine the expression of miR-145 in glucose-treated HUVECs after 6 h. Meanwhile, the gene expression in HUVECs treated with 30 mmol/L of glucose was further examined at different action times (0, 0.5, 1, 2, and 4 h).
For the establishment of IR cell models, HUVECs were treated with insulin of 1, 2, 3, 4, 5 μmol/L on the basis of high-glucose (33.3 mmol/L). HUVECs co-treated with insulin (4 μmol/L) and high-glucose (33.3 mmol/L) were considered as IR models, cells only treated with high-glucose were used as a single comparison, and untreated cells were taken as a blank control. After 6 h of intervention with insulin of different concentrations, glucose consumptions in HUVECs were detected by glucose oxidase-peroxidase method. In controls and IR-treated cells, intracellular glycogen contents of cells were respectively assessed after intervention for 24 and 48 h through anthrone-sulfuric acid method; the morphology of cells after 24 h of treatment were observed with the Eclipse TS-100 inverted microscope (Nikon, Tokyo, Japan). Moreover, the expression of miR-145 in controls, IR-treated and glucose-treated cells were measured via qRT-PCR assay.
Cell transfection
For further exploring the effects of miR-145 on IR-induced HUVECs, miR-145 mimics (#MCH01320, Applied Biological Materials, Vancouver, Canada) and miR-145 inhibitor (#MIH01320, Applied Biological Materials, Vancouver, Canada) were severally transfected into HUVECs (3 × 105) with or without IR. Lipofectamine 2000 (Invitrogen, California, USA) was used for cell transfection according to the manufacturer's protocol. For comparison, un-transfected HUVECs were used as controls, cells transfected with scrambled sequence were served as a negative comparison (NC), and cells only treated with IR were considered as models. Transfected cells were prepared for following experiments, including qRT-PCR, glucose consumption, glycogen content, flow cytometry and western blot (WB) analysis.
qRT-PCR assay
The total RNA of cells was isolated by the Trizol reagents (Invitrogen, Carlsbad, California, USA). The concentration and integrity of isolated RNA was quantified via a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) and 1% agarose modified gel electrophoresis, respectively. The cDNA was synthesized from the 1 µg of isolated RNA with the PrimeScript RT Master Mix Perfect Real Time (TaKaRa, Shiga, Japan) based on the manufacturer’s instructions. The Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) was utilized to ascertain miR-145 expression following the sets: pre-degeneration for 5 min at 95°C, and 35 cycles of 15 s at 95°C and 30 s at 60°C. U6 or GAPDH acted as the internal control, and 2-ΔΔCt method [21] was employed to evaluate miR-145 expression. Sequence: The sequences of primers were shown in Table 1 and synthesized by Gene Pharma (Shanghai, China).
Glucose consumption detection
The glucose oxidase-peroxidase reagent (Robio, Shanghai, China) was used to appraise the glucose consumption of insulin-treated HUVECs and transfected cells. The absorbance at wavelength of 500 nm was detected by Elx-800 microplate reader (Bio-Tek, Vermont, USA), and the glucose in culture was calculated according to the manufacturer's descriptions.
Intracellular glycogen content
The glycogen contents of HUVECs after transfection and insulin treatment were measured by anthranone-sulfuric acid method (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Following the manufacturer's instructions, lye was added into treated HUVECs for boiling water bath for 20 min, and then the developer was instilled into cells for boil water bath for another 5 min. Finally, the absorbance was measured by a landscape photometer (NP80, Implen Gmb H, Germany) at the wavelength of 620 nm, and the glycogen contents of cells were analyzed.
Cell apoptosis
The apoptotic rates of HUVECs after transfection were ascertained by the flow cytometry analysis. For the detection, transfected cells were stabilized in 70% ice-cold ethanol for 1 h, and then stained with 10 μL Annexin V and 5 μL propidium iodide (PI). The apoptotic rates of cells were evaluated using the FACSCalibur flow cytometer (BD Biosciences, USA) following the manufacturer’s instructions.
WB analysis
The expressions of apoptosis-related proteins (Bcl-2 and Bax) in transfected HUVECs were analyzed by WB assays. For the assays, the total protein from cells was isolated using RIPA buffer (Solarbio, Beijing, China). The concentration of total protein was determined through the Bicinchoninic Protein Assay kit (BCA, Pierce, Rockford, IL, USA). Next, the total protein (50 μg) were separated onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China) and then shifted onto polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked with 5% non-fat dried milk for 2 h. The primary antibodies at dilution rate of 1:1000 were added overnight at 4°C, including Bcl-2 (ab59348, Abcam, USA), Bax (ab32503, Abcam, USA), and GAPDH (ab8245, Abcam, USA) acted as the internal reference. The corresponding secondary antibodies goat anti-rabbit IgG H&L (HRP; 1:7000, ab97051, Abcam, USA) and goat anti-mouse IgG H&L (HRP; 1:1000, ab150113, Abcam) were added for 1 h at room temperature. The blots signals were developed by an enhanced chemiluminescence-detecting kit (Thermo Fisher, MA, USA), and the results were normalized to GAPDH.
Statistical analysis
Statistical Package of the Social Sciences 20.0 software (SPSS, Inc., Chicago, USA) was used for data analysis. The measurement data were presented as mean ± standard deviation (SD). The comparison between groups was performed by Student's t-test or one-way analysis of variance (ANOVA). All experiments were carried out in triplicate. P <0.05 was considered as statistically significant.