Ethnic and study participants
This study was approved by the Ethics Committee of Yidu Central Hospital of Weifang, and each participant gave fully informed oral and written consent.
108 patients with asymptomatic AS and 72 healthy controls from Yidu Central Hospital of Weifang were enrolled in this study, all participants had no history of smoke. The asymptomatic AS was diagnosed according to the 2007 Guidelines for the management of arterial hypertension released by the Task Force for the Management of Arterial Hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC)[17]. Patients who had a history of cardiovascular and cerebrovascular diseases, diabetes mellitus, cancer, rheumatic immune disease, acute infection disease, or on medication were excluded. The fasting venous blood was drawn for determining plasma total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and C-reactive protein (CRP). The blood samples were isolated by centrifugation and stored at -80℃ for further analyses. The physical examination was performed for each participant, and the results were recorded. ATL HDI 3000 ultrasound system (Advanced Technology Laboratories, Bothell, WA, USA) was used for the measurement of Carotid intima-media thickness (CIMT).
Cell culture and transfection
Human VSMCs were purchased from the American Type Culture Collection (ATCC). Cells were grown in the Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, GE Health Care, USA) with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, USA). Then cells were incubated in a humidified atmosphere with 5% CO2 at 37℃.
MiR-183-5p agomir, miR-183-5p antagomir, and their relative control miRNAs (agomir NC and antagomir NC) were provided by Ribobio (Guangzhou, China). The sequences were as follows: miR-183-5p agomirs, 5'-UAUGGCACUGGUAGAAUUCACU-3'; miR-183-5p antagomir, 5'-AGUGAAUUCUACCAGUGCCAUA-3'; agomirs NC, 5’-UGUAGGGCCACUCAGUCAACUU-3’; antagomir NC, 5’-AAUAUGGGCGAAAUGGGGCCAUC-3’. As previous study described, cells were transfected with 100 nM miR-183-5p agomir or antagomir, or agomir NC, antagomir NC to regulate the expression level of miR-183-5p by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions[18]. 48h post-transfection, cells were collected for further experiments. To investigate ox-LDL regulation on miR-183-5p expression, VSMCs were treated with ox-LDL (40 μg/ml).
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNAs were isolated from serum or cells by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by purification with Ultrapure RNA Kit (Cwbio, Beijing, China) according to the instructions. Mir-X miRNA First-Strand Synthesis Kit (Takara, Tokyo, Japan) was applied to reverse transcribe RNA. The RT primer sequences were as follows: miR-183-5p, 5'- CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGTGAATT-3'. qRT-PCR was performed using SYBR green I Master Mix Kit (Invitrogen, Carlsbad, CA, USA). U6 was used as an internal control due to its stability in many conditions[19, 20]. The relative expression of the mature miR-183-5p was normalized to U6 based on 2−ΔΔCt method. The PCR primer sequences were as follows: 5'-CGCGCTAT GGCACTGGTAG-3' (forward) and 5'-GTGCAGGGTCC GAGGT-3' (reverse) for miR-183-5p; and 5'-GCGCGTCGTG AAGCGTTC-3' (forward) and 5'-GTGCAGGGTCCGAGGT-3' (reverse) for U6.
Cell proliferation assay
The cell viability was examined using CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instruction. The stably transfected cells were seeded in 96-well plates at a density of 5×104 cells per well. At the indicated time points (0h, 24h, 48h, 72h), 10μl CCK-8 solution was added to each well. The cell viability was reflected by measuring the optical density at the 450 nm using a microplate reader (ELx800, Bio-Tek Instruments, Winooski, VT, USA).
Cell migration assay
For cell migration assay, transwell chamber (Corning, USA) was used. Cells (2 × 104 cells/well) were seeded in chambers and cultured in serum-free DMEM, while 10% FBS was given to the lower chamber as the attractant. Following 48 h of incubation at 37℃, the migrated cells on the lower chamber were stained with 0.1% crystal violet at room temperature for 20 min. Then the number of the migrated cells was counted under an inverted microscope (Olympus Corporation, Tokyo, Japan) using five random fields of view.
Statistical analysis
Statistical analyses were performed using SPSS 21.0 software (SPSS Inc., Chicago, IL) and GraphPad Prism 7.0 software (GraphPad Software, Inc., USA). Data were expressed as the mean ± standard deviation. Comparisons between two groups were performed using student’s t-test, and comparisons between multiple groups were analyzed by one-way ANOVA analysis with a Tukey's post-hoc test. Correlations between continuous variables were assessed using the Person correlation coefficient. The diagnostic value of miR-183-5p for AS patients was assessed by a receiver operating characteristic (ROC) analysis. The area under the curve (AUC) was determined to assess the diagnostic power of the predictors, the larger the AUC, the better the prediction model. AUC = 0.5 indicates no predictive power, whereas AUC = 1 represents perfect predictive performance. P < 0.05 was considered to indicate a statistically significant difference. Each experiment was performed in triplicate.