hPASMCs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in smooth muscle cell medium (ScienCell Research Laboratories, Inc.) supplemented with 2% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin, 100 µg/ml streptomycin and 1% smooth muscle cell growth supplement (all ScienCell Research Laboratories) in an atmosphere containing 5% CO₂ at 37°C. The culture medium was replaced every 2 to 3 days until cell 80% confluence was reached. hPASMCs at passages 4–10 were used in the following experimental assays. Smooth muscle cells were confirmed by immunohistochemistry using anti-α-actin monoclonal antibody (dilution 1:300; sc-130616; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) incubated at 37°C for 2 h. The cells were cultured at 37°C in hypoxic conditions (5% CO₂ and 5% O₂) or normoxic conditions (1% CO₂ and 20% O₂) as control for 6, 12 and 24 h respectively, prior to the experiment. Subsequently, after treatment with mitoK_ATP inhibitor 5-hydroxydecanoate (5-HD; 500 µmol/l), mitoK_ATP agonist diazoxide (100 µmol/l) or HIF-1α inhibitor 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1; 50 µmol/l; all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or smooth muscle cell medium supplemented with 2% fetal bovine serum as control for 1 h, immediately the cells were further maintained at 37°C in a humidified atmosphere of 5% CO₂ and 5% O₂ for 6,12 and 24 h.

mRNA and miRNA PCR experiments were performed in triplicate. For mRNA expression assay, total RNA was extracted from treated cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). cDNA was synthesized using a ReverTra Ace qPCR RT kit (Toyobo Co., Ltd., Osaka, Japan). RNA concentration and purity were assessed by UV spectrophotometry (1.8<A260/A280<2.0). RNA integrity was assessed using electrophoresis. Reverse transcription reaction was performed using 4 µg of total RNA and the First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. To generate standard curves, 1 µl of first-strand cDNA was amplified using a Premix Ex Taq Version Kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's instructions and quantification of PCR products was assessed to plot standard curves. The qPCR reactions were performed using a SYBR Green PCR Master mix (CWBIO; Beijing, China) in a CFX-96 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the following primers: HIF-1α, forward 5′-CTGATCATCTGACCAAAACTC-3′ and reverse 5′-GTTTCAACCCAGACATATCCAC-3′; and β-actin, forward 5′-ACTCTTCCAGCCTTCCTTCC-3′ and reverse 5′-CGTCATACTCCTGCTTGCTG-3′. Real-time PCR was performed using the SYBR Premix Ex TaqTM II (Takara Bio, Inc.) protocol on a Bio-Rad Connect real-time PCR detection system with cycling conditions of 95°C for 3 min, followed by 40 cycles of 95°C for 30 sec and 60°C for 30 sec. β-actin was used as an internal control. The relative mRNA expression levels were determined using the 2^−ΔΔCq method and normalized to β-actin mRNA (12,13). For the miR-210 expression assay, total cellular RNA was extracted with TRIzol reagent. cDNA was synthesized by reverse transcription using the miRNART-primer (RibBio Co., Ltd., Guangzhou, China). The qPCR reactions were performed using a SYBR Green PCR Master mix using miR-210 and U6 primer purchased from RibBio Co., Ltd. (sequences not supplied), which served as normalization control. qPCR was performed under the following steps: 95°C for 3 min, followed by 40 cycles of 95°C for 30 sec and 56°C for 30 sec. miR-210 expression relative to U6 control was calculated using the 2^−ΔΔCq method (14).

The ΔΨm was monitored using flow cytometry according to a modified method (15). Briefly, treated cells were detached by trypsinization and washed twice in PBS. A total of 10 µg/ml rhodamine-123 (R-123, Sigma-Aldrich; Merck KGaA) was then added and incubated for 30 min at 37°C in the dark. The fluorescence intensity of cells was measured using a FACSAria II cytometer (BD643182; BD Biosciences, San Jose, CA, USA).

Measurement of mitochondrial intracellular ROS was performed as described previously (11). The fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was purchased from Sigma-Aldrich; Merck KGaA.

A total of 2×10⁵ cells were plated into 6-well plates ins mooth muscle cell medium supplemented with 2% fetal bovine serum in normoxic conditions (1% CO₂ and 20% O₂) at 37°C for 24 h. Cells were transfected with miR-210 mimic (miR-210) or inhibitor (anti-210) (RibBio Co., Ltd.) at a final concentration of 80 nmol/l, or three small interfering RNAs (siRNA1, siRNA2 and siRNA3) against ISCU (GenePharma, Shanghai, China) at a final concentration of 5 nmol/l using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) with serum-free smooth muscle cell medium, as described previously (11). The follow sequences were used: miR-210 mimic, forward 5′-CUGUGCGUGUGACAGCGGGUGA-3′ and reverse 5′-UUGACACGCACACUGUCGCCGA-3′; and inhibitor, forward 5′-UCAGCCGCUGUCACACGCACAG-3′ and reverse 5′-CAGUACUUUUGUGUAGUACAA-3′. After 6 h, the culture medium was replenished with fresh smooth muscle cell medium supplemented 2% fetal bovine serum and cells were cultured for an additional 24 h at 37°C, then subjected to hypoxia (5% CO₂ and 5% O₂) and normoxia (1% CO₂ and 20% O₂) at 37°C for 24 h. Control miR sequences and control siRNA sequences were used (miR-con, anti-con, si-con) as negative control. After miR-210 inhibitor and control transfection for 24 h, cells were treated with 5-HD (500 µmol/l) or diazoxide (100 µmol/l) and exposed to hypoxia for 48 h at 37°C.

Cells were lysed with lysis buffer (1% Triton X-100, 50 mmol/l Tris-HCl, pH 7.4, 25 mmol/l glycerophosphate, 150 mmol/l NaCl, 2 mmol/l EDTA, 2 mmol/l ethylene glycol tetraacetic acid, 1 mmol/l phenylmethylsulfonyl fluoride, 10% glycerol and 1% protease and phosphatase inhibitors) for 10 min on ice. The lysates were collected and centrifuged at 15,000 × g for 15 min at 4°C. The supernatant was recovered, and protein concentration was measured using a protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). The proteins loaded (40 mg per lane) were subsequently separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (PVDF; Thermo Fisher Scientific, Inc.), blocked at 37°C for 2 h in 5% non-fat milk, and incubated with anti- proliferating cell nuclear antigen (PCNA; sc-25280) or anti-ISCU antibody (sc-271536) or β-actin antibody (sc-47778) or GAPDH (sc-25778) at 1:4,000 or 1:3,000 or 1:10,000 or 1:5,000, respectively (Santa Cruz Biotechnology, Inc.) at 4°C overnight. After washing for 10 min in 1X Tris-buffered saline-Tween-20 solution three times, the target protein was probed with the horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (sc-3841; at 1:6,000 dilution; Santa Cruz Biotechnology, Inc.) at 37°C for 2 h. After washing for 10 min in 1X Tris-buffered saline-Tween-20 solution three times, the bound antibody on the PVDF membrane was detected by chemiluminescence with the ECL Detection Reagent kit (Pierce; Thermo Fisher Scientific, Inc.) and the protein expression was analyzed using Quantity One 4.6.2 (Bio-Rad Laboratories, Inc.) (16).

Statistical analysis was performed using one-way analysis of variance and Duncan's test using Graphpad software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA). The data were expressed as the mean ± standard deviation in three independent experiments. The statistical significance of differences among groups was determined using an analysis of variance or Student's t-test. P<0.05 was considered to represent a statistically significant difference.

Hypoxia-triggered mitoK_ATP channel opening initiated by ATP depletion leads to the collapse of ΔΨm, which has been implicated in the generation of ROS (17). Consistent with a previous report (11), the present study observed that R-123 fluorescence intensity was significantly increased in hPASMCs subjected to hypoxia for 12 and 24 h (P<0.05 and P<0.01, respectively), relative to normoxic control cells (Fig. 1A), indicating that ΔΨm depolarization had occurred. Furthermore, hPASMCs exhibited significantly increased levels of ROS after 12 and 24 h of hypoxia (P<0.05), as determined by DCFH-DA staining (Fig. 1B). To further investigate whether mitoK_ATP channels were associated with ROS production in hPASMCs, the levels of intracellular ROS were measured following treatment with diazoxide or 5-HD, to activate or inhibit mitoK_ATP, respectively. As predicted, opening of mitoK_ATP significantly increased the level of intracellular ROS, and closure of mitoK_ATP significantly decreased the level of intracellular ROS under hypoxic conditions (P<0.05; Fig. 1C). Furthermore, under normoxic conditions, the opening of mitoK_ATP significantly increased the level of intracellular ROS while closure of mitoK_ATP significantly decreased the level of intracellular ROS (P<0.05; Fig. 1C). These results suggest that mitoK_ATP channels may be involved in the generation of ROS in hPASMCs under hypoxic conditions, potentially through the collapse of ΔΨm.

As HIF-1α is a critical oxygen sensor and master transcriptional regulator of the hypoxic adaptive response, it has been suggested that mitoK_ATP channels function in combination with HIF-1α and its downstream targets, including miR-210 and ISCU (12,18). To investigate whether mitoK_ATP was associated with the expression of HIF-1α and miR-210 in hPASMCs during hypoxia, the expression of HIF-1α mRNA and miR-210 was measured using RT-qPCR. In hPASMCs subjected to hypoxia, levels of HIF-1α mRNA were significantly increased at 12 and 24 h (P<0.01 and P<0.001, respectively), and miR-210 levels were significantly increased at 6 and 12 h (P<0.05; Fig. 2A). In turn, expression of miR-210 was significantly decreased following treatment with the HIF-1α inhibitor YC-1 (P<0.01; Fig. 2B), and expression of HIF-1α was significantly increased following treatment with the mitoK_ATP agonist diazoxide (P<0.05; Fig. 2C). Furthermore, the levels of HIF-1α mRNA and miR-210 were significantly decreased following closure of mitoK_ATP with 5-HD (P<0.001 and P<0.01, respectively; Fig. 2C). To further investigate whether HIF-1α was involved in the regulation of mitoK_ATP, ΔΨm and ROS levels were measured in hPASMCs treated with YC-1. Significant decreases in the fluorescence intensity of R-123 (P<0.05) and DCFH-DA (P<0.01) were observed in hPASMCs upon inhibition of HIF-1α (Fig. 2D).

These data suggest that mitoK_ATP channel opening increases the expression of HIF-1α and its downstream target miR-210, leading to HIF-1α-mediated regulation of mitoK_ATP through increased ROS levels in a positive feedback manner in hPASMCs under hypoxic conditions.

Previous studies have indicated that ISCU is a downstream regulatory target of miR-210 (19,20). ISCU, as an essential component of respiratory electron transfer complexes, is involved in ROS generation in the mitochondria (21). The present study blocked the expression of miR-210 using an inhibitor of miR-210. Under normoxic and hypoxic conditions, significant decreases in the fluorescence intensity of R-123 (P<0.001 and P<0.01, respectively) and DCFH-DA (P<0.01) were observed in hPASMCs upon treatment with miR-210 inhibitor transfection (Fig. 3A and B), indicating an involvement of miR-210 in the alteration of ΔΨm and generation of ROS. The expression of ISCU was subsequently assessed, and was demonstrated to be reduced or increased following treatment with miR-210 mimic or miR-210 inhibitor under normoxic conditions, respectively (Fig. 3C). As shown in Fig. 3C, ISCU expression was reduced under hypoxic conditions compared with normoxic conditions.

Furthermore, in order to determine the role of ISCU in the activity of mitoK_ATP, specific siRNA against ISCU or scrambled control siRNA was transfected into hPASMCs. Western blot analysis indicated that two of the three siRNAs specifically reduced the expression of ISCU (Fig. 3D). In addition, a significant increase in the fluorescence intensity of R-123 was observed in hPASMCs upon treatment with ISCU siRNA (P<0.01; Fig. 3E), which suggests a role of ISCU in the collapse of ΔΨm, the fluorescence intensity of R-123 was lower in hypoxic environments. Loss of ISCU may impair the function of the electron transfer chain, leading to increased generation of ROS and decreased production of ATP. Decreased levels of ATP may then trigger opening of mitoK_ATP channels and a collapse of ΔΨm.

These data suggest that mitoK_ATP channels indirectly participate in ROS generation in hPASMCs under hypoxic conditions via the miR-210/ISCU pathway. This may supplement the potential direct involvement of mitoK_ATP in ROS generation, mediated by channel opening and ΔΨm collapse.

As hPASMCs grow relatively slowly, the proliferation of hPASMCs was evaluated by measuring the expression of PCNA, as an established marker of cell proliferation that is synthesized in the early G1 and S phases of the cell cycle (22). Results of western blot analysis showed that PCNA expression was markedly increased in hPASMCs subjected to hypoxia for 48 h, relative to normoxic controls (Fig. 4A). The proliferation of hPASMCs upon activation or inhibition of mitoK_ATP and inhibition of miR-210 was subsequently assessed. It was observed that activation of mitoK_ATP increased the expression of PCNA, while inhibition of mitoK_ATP and miR-210 decreased PCNA expression (Fig. 4B). These results indicate that mitoK_ATP promotes the proliferation of hPASMCs under hypoxic conditions through the miR-210 signaling pathway.