Baicalin (purity, >99.0%; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in dimethylsulfoxide. The antibodies against PCNA (cat. no. 10205-2-AP; 1:1,000), cyclin A (cat. no. 13295-1-AP; 1:2,000), cyclin E (cat. no. 11554-1-AP; 1:2,000) and β-actin (cat. no. 60008-1-1g; 1:5,000) were purchased from ProteinTech Group, Inc. (Chicago, IL, USA). CycleTEST™ PLUS DNA Reagent kit was obtained from BD Biosciences (Franklin Lakes, NJ, USA). The bromodeoxyuridine (BrdU) proliferation kit was purchased from EMD Millipore (Billerica, MA, USA). Ox-LDL, VicFemto enhanced chemiluminesence (ECL) kit and PBS were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China).

The present study was approved by the Ethics Committee for the use of human samples of the First People’s Hospital of Lianyungang, which was in accordance with the code of ethics of the Declaration of Helsinki developed by the World Medical Association. Each individual provided written informed consent prior to their participation. Serum samples were collected from 40 patients with AS (age, 60±10 years; 18 male and 22 female) and 40 healthy volunteers (age, 58±9 years; 21 male and 19 female) from the Department of Cardiovascular Medicine, the First People’s Hospital of Lianyungang (Lianyungang, China) between January 2015 and January 2016.

The AS group included patients with >80% coronary artery stenosis confirmed by coronary angiogram. The control group included patients without clinically significant coronary artery occlusion diagnosed by coronary angiogram. Patients with vascular diseases, including hypertension and type I/II diabetes were excluded. Total RNA was extracted from peripheral blood monocytes of patients and controls.

HA-VSMCs were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in F-12K medium (American Type Culture Collection) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Other nutrients added to the HA-VSMC culture were based on previously published literature (27). Cells were incubated at 37˚C with 5% CO₂.

The sequences of siRNA targeting lncRNA MEG3 and scrambled negative control siRNA (si-NC) were designed and synthesized by GenePharma (Shanghai GenePharma Co., Ltd., Shanghai, China) according to the previously published protocol (15). pcDNA-MEG3 and empty vector (pcDNA) were synthesized by GenePharma (Shanghai GenePharma Co., Ltd.). At 60% confluence, HA-VSMCs were cultured in Dulbecco’s modified Eagle’s medium (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂ for 24 h. HA-VSMCs were transfected with si-NC, si-MEG3 at a final concentration of 20 nmol/l in accordance with the manufacturer’s protocol. Furthermore, HA-VSMCs were transfected with pcDNA-MEG3 at a final concentration of 2 mg/l using. The following sequences were used: si-lncRNA MEG3, 5′-GGGCTTCTGGAATGAGCAT-3′; si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′ (15). HA-VSMCs were transfected using Lipofectamine^® 2000 on 6-well plates (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. Cells were harvested for the subsequent experiments 24 h after transfection.

Cell proliferation analysis was performed with BrdU. Transfected and untransfected cells were seeded into 96-well plates at a density of 1×10⁴ cells/well and treated with control (no treatment) and different concentrations of ox-LDL (25, 50 or 75 µg/ml) and baicalin (5, 10 or 20 µmol/l) in DMEM containing 2% FBS at 37°C with 5% CO₂ and BrdU was added to the proliferating cells 2-24 h before the end of the test reagent incubation. The following steps were performed according to the manufacturer’s protocol and as previously described (28). Finally, the absorbance was measured at a dual wavelength of 450/550 nm.

Total RNA was isolated from serum samples and HA-VSMCs using the TRIzol reagent according to the manufacturer’s protocol (Thermo Fisher Scientific, Inc.). RT reaction and qPCR were performed as previously described (15). cDNA was reverse transcribed from 100 ng of total RNA using 4 µl of miScript HiSpec buffer, 2 µl of Nucleics Mix, 2 µl of miScript Reverse Transcriptase mix (Qiagen GmbH, Hilden,

Germany) and sterilized distilled water up to a total volume of 20 µl. The RT reaction was performed at 37°C for 60 min followed by heat inactivation at 95°C for 5 min. The qPCR reactions were performed in triplicate using 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Reaction mixtures had a total volume of 25 µl, including 2 µl of cDNA, 12.5 µl of QuantiTect SYBR-Green PCR master mix (Qiagen GmbH), 2.5 µl of universal primers (Qiagen GmbH), 2.5 µl of MEG3 or GAPDH-specific primers and 5.5 µl of nuclease-free water. Reactions were incubated in 96-well optical plates (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the following thermocycling conditions: Initial denaturation at 95°C for 10 min; 40 cycles of 95°C for 10 sec and 60°C for 30 sec. At the end of the PCR cycles, melting curve analysis was performed to validate the specific generation of the expected PCR products. Primers were designed using Applied Biosystems Primer Express 3.0 (Applied Biosystems; Thermo Fisher Scientific, Inc). GAPDH was used as internal reference and the 2^−ΔΔCq method was used to calculate the expression of lncRNA MEG3 in samples from serum and cells (29). The following primer sequences were used for the qPCR: MEG3: 5′-CTGCCCATCTACACCTCACG-3′ (forward) and 5′-CTCTCCGCCGTCTGCGCTAGG GGCT-3′ (reverse); GAPDH: 5′-GAAGGTGAAGGTCGGAGTC-3′ (forward) and 5′-GAAGATGGTGATGGGATTTC-3′ (reverse).

A total of 24 h after transfection, cells were washed 3 times with PBS and incubated with radioimmunoprecipitation assay lysis buffer (cat. no. KGP702-100; Nanjing KeyGen Biotech Co., Ltd.) for 30 min on ice. After 30 min of cell lysis, cell lysates were sonicated with 20 KHz frequency on ice for 1 min and centrifuged at 14,000 × g for 15 min at 4°C, and the protein concentration was quantified using bicinchoninic acid protein concentration kit (Nanjing KeyGen Biotech Co., Ltd.). Samples containing 20 µg total protein were analyzed using SDS-PAGE (10% gel) using an electrophoresis instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and western blotting was performed according to the previously published protocol (28). Antibodies against PCNA, cyclin A and E, p53 and β-actin were used overnight at 4°C and washed 6 times with tris buffered saline with Tween-20 (TBST) at room temperature (5 min/wash). Subsequently, the membrane was treated with horseradish peroxidase labeled goat-anti-rabbit immunoglobulin G (IgG) secondary antibody (cat. no. VA002; 1:5,000; Xuzhou VICMED Biological Technology Co., Ltd., Xuzhou, China; www.vicmed.cn) or goat-anti-mouse IgG secondary antibody (cat. no. VA001; 1:5,000; Xuzhou VICMED Biological Technology Co., Ltd.), shaken and incubated at room temperature for 1 h. Following washing with TBST 6 times at room temperature (5 min/wash), the membranes were incubated with ECL solution for 5 min. Bands were imaged using VersaDoc™ MP 4000 (Bio-Rad Laboratories, Inc.) and analyzed by using PDQuest Advanced 2D analysis software (version 8.0, Bio-Rad Laboratories, Inc.).

The transfected HA-VSMCs were cultured in 6-well plates. To perform cell cycle analysis, the cells were digested with trypsin and fixed with 70% ethanol for 24 h at 4°C. The cells were stained according to the manufacturer’s protocol of CycleTEST PLUS DNA Reagent kit (BD Biosciences). The fixed cells were washed 3 times with PBS and centrifuged at 300 × g for 5 min in room temperature. The supernatant was subsequently discarded. Cells were incubated with propidium iodide (PI) stain buffer for 10 min and subsequently treated with 200 µl of PBS, 200 µl of RNase A and 200 µl of PI for 10 min at 4°C in the dark. Finally, the cells were screened by 400-mesh sieves and analyzed by flow cytometry (FACSCanto II; BD Biosciences). The results obtained were analyzed using the ModFit software (version 4.1; Verity Software House, Inc., Topsham, ME, USA).

For the wound healing assay, the following groups of HA-VSMCs were cultured in 6-wellplates until 60% confluence: control, si-NC, si-lncRNA MEG3, si-lncRNA MEG3 + baicalin (5 µmol/l), si-lncRNA MEG3 + baicalin (10 µmol/l), si-lncRNA MEG3 + baicalin (20 µmol/l). Cells were scratched with pipette tips and washed three times with PBS (15). Images of scratch areas were captured at 0 and 24 h. Each experiment was repeated five times independently.

The potential of mitochondrial membrane was analyzed as previously described using JC-1 staining (30). Mitochondrial depolarization is demonstrated by increased green/red fluorescence intensity ratio. When mitochondrial membrane potential level is low, green fluorescence is primarily observed. When the mitochondrial membrane potential increases, a polymer is formed and red fluorescence is observed (31). Mitochondrial membrane potentials were detected using a fluorescent microscope with 488 nm excitation wavelength.

Cell proliferation was analyzed using kFluor647 Click-iT EdU kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer’s protocol. HA-VSMCs were seeded into 96-well cell culture plates at a density of 1×10⁴ cells/well. Following transfection and treatment with baicalin, HA-VSMCs were incubated with 10 µmol/l EdU for 4 h at 37°C. Cells were permeabilized for 20 min with 0.5% Triton X-100 after fixing with 4% formaldehyde for 30 min at room temperature. Cells in each well were washed three times with 0.1 ml 3% bovine serum albumin (cat. no. VIC018; Xuzhou VICMED Biological Technology Co., Ltd.) in PBS and subsequently incubated with 1X Click-iT EdU reaction buffer at room temperature for 30 min in the dark. HA-VSMC nuclei stained by Hoechst were used to count cells and visualization was performed using a fluorescence microscope as described below (Nikon Corporation, Tokyo, Japan).

Treated HA-VSMCs were cultured on 24-well cell culture plates. Subsequently, 1 µl of Hoechst 33342 (5 mg/ml; Sigma-Aldrich; Merck KGaA) in 200 µl of PBS was added to HA-VSMCs and incubated for 20 min in room temperature. The stained cells were washed three times with PBS, and cell morphology was observed under a fluorescence microscope.

HA-VSMCs were fixed with 4% paraformaldehyde for 15 min at room temperature, and washed in cold PBS 3 times for 5 min. Permeabilization was performed in 0.4% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature. For immunofluorescence staining, the experiment was conducted as previously described (15).

Cell viability was assayed by the MTT assay, as previously described (28). HA-VSMCs were cultured in 96-well microtitration plates and then the cells were subjected to growth arrest in DMEM without serum for 24 h. Following transfection and treatment, HA-VSMCs were incubated for 4 h in a medium containing 0.5% MTT. Thereafter, the supernatant was removed, and dimethylsulfoxide (150 µl/well) was added. The plates were then agitated on a plate shaker for 10 min at room temperature. The absorbance was measured at a wavelength of 490 nm in a spectrophotometer.

Data are presented as the mean ± standard error of the mean. Comparisons between two groups were performed using unpaired Student’s t-test and three or more groups were compared using one-way analysis of variance, followed by Dunnett’s test. P<0.05 was considered to indicate a statistically significant difference.

The current study determined the expression levels of MEG3 in serum samples from patients with AS and HA-VSMCs treated with ox-LDL. RT-qPCR assay indicated that the expression level of MEG3 significantly decreased in patients with AS compared with healthy controls (Fig. 2A). A previous study reported that ox-LDL serves an important role in the progression of AS (32). Ox-LDL promotes osteogenic differentiation and proliferation of VSMCs (33), and has been widely used to induce a model of AS (27,32,33). Consistent with these results, the current study indicated that ox-LDL promoted cell proliferation in a concentration dependent manner (Fig. 2B and C). In addition, the expression levels of MEG3 were detected in HA-VSMCs after treatment with ox-LDL. The expression of MEG3 decreased following stimulation with ox-LDL. The results indicated that the expression level of MEG3 decreased in a concentration-dependent manner. The greatest inhibition of MEG3 expression was achieved using a dose of 75 µg/ml ox-LDL, and, therefore, this dose was used in subsequent experiments (Fig. 2D). These results indicated that MEG3 may be a mediator in AS progression. HA-VSMCs treated with ox-LDL were used as an in vitro model of AS and subsequently treated with baicalin. The present study aimed to determine whether different concentrations of baicalin affected the expression of MEG3. Data indicated that different concentrations of baicalin did not affect the expression of MEG3 in the absence of oxLDL treatment (Fig. 2E), suggesting that this drug did not affect the expression of MEG3 in normal cells. However, in ox-LDL-treated HA-VSMCs, incubation with ox-LDL resulted in a significant decrease in MEG3 expression compared with the control. Baicalin reversed this effect in a dose-dependent manner (Fig. 2F).

To verify the function of MEG3 in the progression of AS, the current study determined the effects of MEG3 silencing on proliferation of ox-LDL-treated HA-VSMCs. HA-VSMCs treated with 75 µg/ml ox-LDL were transfected with si-MEG3 and treated with different concentration of baicalin (5, 10 and 20 µmol/l), followed by measurement of cell proliferative ability. The knockdown of MEG3 was confirmed by qPCR (Fig. 3A). BrdU assay indicated that downregulation of MEG3 caused a significant increase in cell proliferation compared with the si-NC group in ox-LDL-treated HA-VSMCs. However, cell proliferation was inhibited following treatment with different concentrations of baicalin (Fig. 3B). To determine whether baicalin affected cell cycle progression by increasing MEG3 expression in the presence of ox-LDL, flow cytometry was used to detect the number of cells at different stages of the cell cycle. MEG3 knockdown increased the percentage of cells at the G₂/M+S phase compared with the si-NC group. Baicalin inhibited cell cycle progression and increased the number of HA-VSMCs at the G₀/G₁ phase (Fig. 3C and D). EdU staining analysis indicated that the number of proliferating cells increased following knockdown of MEG3 compared with the si-NC group (Fig. 3E and F). This effect was reversed in the presence of baicalin at a concentration of 10 or 20 µmol/l. MEG3 is a tumor suppressor gene, and its ectopic expression inhibited cell proliferation and promoted cell apoptosis in human glioma cell line (34). Our previous study indicated that MEG3 knockdown may trigger human pulmonary artery smooth muscle cell proliferation and migration (15). In the present study, MEG3 knockdown promoted the proliferation of HA-VSMCs in the absence of ox-LDL, compared with the si-NC group. However, compared with the MEG3 knockdown group untreated with ox-LDL, proliferation increased in cells transfected with si-lncRNA MEG3 and treated with ox-LDL (Fig. 4A). In the absence of ox-LDL, baicalin inhibited the proliferation of HA-VSMCs transfected with si-lncRNA (Fig. 4B). The above results indicated that baicalin inhibited the proliferation induced by MEG3 knockdown and stimulation with ox-LDL. Furthermore, RT-qPCR analysis of MEG3 expression was performed following transfection with pCDNA-MEG-3. Compared with the pCDNA group, the expression level of MEG3 increased by 1.59-fold in the pCDNA-MEG3 group (Fig. 5A). Furthermore, overexpression of MEG3 suppressed the viability and proliferation in ox-LDL-treated HA-VSMCs compared with the ox-LDL + pCDNA group (Fig. 5B and C).

It has been reported that migration of HA-VSMCs contributed to vascular remodeling in AS (35). The current study examined the effect of baicalin on MEG3 silencing-induced migration of HA-VSMCs using a wound healing assay. Ox-LDL- treated HA-VSMCs were treated with control, si-NC, si-lncRNA MEG3, si-lncRNA MEG3 + baicalin (5 µmol/l), si-lncRNA MEG3 + baicalin (10 µmol/l) and si-lncRNA MEG3 + baicalin (20 µmol/l). A scratch was made using a pipette tip and images were captured at 0 and 24 h to analyze the degree of migration in different treatment groups. The results indicated that knockdown of MEG3 increased the migration of HA-VSMCs, compared with the si-NC group; however, treatment with baicalin at a dose of 10 and 20 µmol/l significantly reversed the effect of MEG3 knockdown (Fig. 6A and B).

In order to further investigate whether baicalin could promote apoptosis in AS in vitro, the expression of MEG3 was decreased in HA-VSMCs through pre-treatment with ox-LDL. Alterations in mitochondrial function were initially investigated. According to previous studies, disruption of mitochondrial membrane potential is an early event of apop-tosis (31,36,37). The alterations of mitochondrial membrane potential were verified by JC-1 staining. Following MEG3 knockdown the green/red fluorescence ratio was reduced compared with the si-NC group, suggesting an increase in mitochondrial membrane potential. By contrast, the mitochondrial membrane potential decreased following treatment with baicalin, as evidenced by increased ratio of green to red fluorescence (Fig. 7A and B). Furthermore, chromatin condensation and cell nuclear morphology were observed by staining with Hoechst 33342. The MEG3 knockdown group exhibited the same complete nuclear morphology as the NC group in cells, while treatment with baicalin resulted in alterations in chromatin morphology including crenation, condensation and fragmentation (Fig. 7C).

To further determine the mechanism underlying baicalin-mediated inhibition of MEG3 knockdown-induced proliferation, the expression of PCNA was determined by western blotting. The results indicated that MEG3 knockdown increased the expression of PCNA, which was reversed in the presence of different concentration of baicalin (Fig. 8A). Cyclin A and E serve roles in cell cycle progression, and, therefore, expression levels of these proteins were determined in ox-LDL-stimulated HA-VSMCs. The results indicated that knockdown of MEG3 increased the expression of cyclin A and E; however, this effect was reversed by treatment with baicalin (Fig. 8B and C). These results indicated that baicalin may serve a role in regulating cell cycle progression and inhibiting cell proliferation.

Previous studies demonstrated that the p53 signaling pathway serves a role in AS (38,39). To further examine the mechanism underling baicalin-mediated inhibition of cell growth, p53 immunofluorescence and western blotting were performed in ox-LDL-treated HA-VSMCs. Fluorescence data indicated that p53 was expressed in the nucleus under normal conditions; however, cytoplasmic expression was detected following knockdown of MEG3. Following treatment with different concentrations of baicalin, p53 was activated and returned to nuclear expression (Fig. 9A). Western blotting further indicated that the low expression of p53 following MEG3 knockdown gradually increased following administration of different concentrations of baicalin (Fig. 9B). The above data indicated that baicalin inhibited the growth of HA-VSMCs possibly through the p53 signaling pathway.