MicroRNA153 induces apoptosis by targeting NFATc3 to improve vascular remodeling in pulmonary hypertension

ABSTRACT

Background

The present study aimed to investigate the effect of microRNA153 (miRNA153) on pulmonary hypertension (PH).

Methods

PH was induced by a single subcutaneous injection of sugen5416 (SU5416) combined with hypoxia exposure for 3 weeks (SuHx) in rats, while pulmonary arterial smooth muscle cells (PASMCs) obtained from rats were exposed to hypoxia to establish an in vitro model. Through observing the characteristic hemodynamic index in rats and by analyzing the physiological function, vascular remodeling and right ventricular hypertrophy were identified. The regulatory effects of miRNA153 on the nuclear factor of activated T cell isoform c3 (NFATc3) were measured by RT-qPCR, western blot, and immunofluorescence. Cell apoptosis was evaluated by flow cytometry.

Results

The miRNA153 expression was reduced and unclear translation of NFATc3 was increased in both the in vivo and in vitro models of PH. In vivo, the pulmonary arterial pressure, right ventricle/(left ventricle + interventricular septum) (RV/(LV+S)), and media vascular thickness were increased in rats with PH; however, all these parameters were suppressed by prophylactic administration of miRNA153agomir. The upregulation of NFATc3 and downregulation of the potassium voltage-gated channel subfamily A member 5 (Kv1.5) were also reversed by transfection with miRNA153agomir. In vitro, miRNA153 increased the level of Kv1.5 in hypoxic PASMCs by targeting NFATc3 and inhibiting their proliferation and apoptosis resistance.

Conclusion

Our results confirmed that the therapeutic administration of miRNA153 promotes apoptosis and inhibits the proliferation of PASMCs to ameliorate PH, and that the NFATc3/Kv1.5 channel pathway may be involved in this process.

KEYWORDS:

• Pulmonary hypertension
• microRNA153
• pulmonary artery smooth muscle
• apoptosis
• nuclear factor of activated T cell isoform c3

Introduction

Pulmonary hypertension (PH) is a progressive pulmonary destructive disease. In PH, abnormally increased pulmonary arterial pressure and vascular vasoconstriction result in irreversible vascular remodeling and culminate in right heart failure (1).The disorder of pulmonary arterial smooth muscle cells (PASMCs) proliferation/apoptosis balance, which aggravates the decrease in vascular elasticity, is the key to the development of this disease (2,3). PH is a disease with poor survival rates, and in spite of the continuous progress of methods, current treatments do little to alleviate vascular remodeling. Therefore, it is very important to actively explore the underlying pathological mechanism of PH and seek suitable treatment methods.

Nuclear factor of activated T (NFAT) cells, which consist of five members (NFATc1, NFATc2, NFATc3, NFATc4, and NFATc5), was initially identified in activated T cells (4). During hibernation, the NFAT protein resides in the cytoplasm in a phosphorylated state. However, when there is an increase in the intracellular calcium concentration induced by store-operated Ca²⁺ entry, the NFAT protein is dephosphorylated by calcineurin, and it exposes nuclear localization signals that link Ca²⁺ signaling to target genes (5). In smooth muscle, NFAT activity is closely related to Ca²⁺ concentration, and it is involved in the development of cardiovascular diseases by regulating the transcription factor (6). Hypoxia promotes excessive proliferation and resistance to apoptosis in PASMCs. The imbalance of proliferation and apoptosis plays an important role in the occurrence and development of pulmonary arterial remodeling in PH (7,8). A previous study revealed that the nuclear factor of activated T cell isoform c3 (NFATc3) is activated in adult and neonatal mice exposed to chronic hypoxia; however, hypoxia induced PH was absent in NFATc3 deficient mice (9). Another study has shown that carboxyl-terminus of hsc70 interacting protein (CHIP) attenuates lipopolysaccharide (LPS)‐induced cardiac hypertrophy and apoptosis by promoting NFATc3 proteasomal degradation (10). Additionally, it was indicated that NFATc3 deletion attenuated metabolism syndrome, reduced the expression of inflammatory regulators and apoptosis of hypothalamic cells (11). On the contrary, in PASMCs, activation of NFATc3 increased hypoxia-induced apoptosis resistance and aggravated vascular remodeling (12). NFATc3 has specifically been implicated in vasculature development of hypoxia-induced PH. However, the function of NFATc3 in hypoxia-induced imbalance of proliferation and apoptosis remains unknown.

MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that negatively regulate gene expression by degrading or translating target miRNAs, and they act as post-transcriptional regulators, with a length of about 18–25 nucleotides (13,14). Most of the genome is not directly involved in protein synthesis, but in the encoding of non-coding RNA (15). miRNAs are key regulators of a variety of cellular processes, and their discovery has revolutionized our understanding of gene regulation systems. A specific miRNA can target multiple mRNAs; however, an mRNA can be regulated by multiple miRNAs. In addition, various related miRNAs can affect a pathway at different levels (16). The process of vascular remodeling in PH includes injury and repair of endothelial cells and recruitment of PASMCs, and specific miRNAs are involved through targeted regulatory genes. For this reason, miRNAs have been hypothesized as potential therapeutic targets for several types of disease. The RhoA/ROCK signaling pathway is significantly activated in hypoxia-induced PH and is involved in the regulation of vasoconstriction, while microRNA141 can target RhoA to reverse hypoxia-induced proliferation and migration of PASMCs (17) .A recent report showed that miRNA153 significantly inhibited the proliferation and migration, and promoted the apoptosis by targeting AKT in lung cancer (18). Meanwhile, miRNA153 could repress glioblastoma multiforme stem cell line growth and promote apoptosis (19). However, whether miRNA153 is involved in resistance to apoptosis in hypoxic PASMCs is unclear.

As we previously found, the miRNA153 appears to be a potential molecular target of inhibiting proliferation and migration by targeting NFATc3 and ROCK1 in hypoxia-induced human PASMC (20). Based on the research, our study aimed to investigate the role of miRNA153 in sugen5416 (SU5416) combined with hypoxia (SuHx)-induced PH model and hypoxic PASMCs.

Materials and methods

Animals

Adult male Sprague-Dawley (SD) rats weighing 180–220 g were purchased from the SPF Animal Center of Jinzhou Medical University. All rats had free access to food and water and were housed at room temperature (20 ± 2°C) and humidity (50 ± 10%). All our animal experiments were conducted in accordance with the guidelines of the National Animal Society, and they were approved by the Ethics Committee of Jinzhou Medical University and the Animal Protection Association. All experimental operators had obtained the ethical assessment certificate of animal experiments. Animal health and behavior were monitored twice a week.

Rat model of pulmonary hypertension

The hemodynamic parameters and morphology index change are the most obvious, and the mortality is low after 3 weeks of hypoxia combined with treatment of SU5416, as previously described (21,22). Male SD rats were randomly divided into the following 4 groups (n = 10 per group): Normal group (N), SuHx-1 w, SuHx-2 w, and SuHx-3 w. SU5416 (20 mg/kg) combined with hypoxia (10% O₂, 24 hours/day) is named as SuHx (23). SU5416 was suspended in 0.5% (wt/vol) carboxymethylcellulose sodium, 0.9% (wt/vol) NaCl, 0.4% (vol/vol) polysorbate, and 0.9%(vol/vol) benzyl alcohol in dH₂O. For normal group, the rats were housed under normoxia (21% O₂) for 21 days; SuHx-1 w, the rats were housed in normoxic for 14 days followed by the injection of 20 mg/kg SU5416 at day 15 and hypoxia from day 15 to day 21; SuHx-2 w, the rats were exposed in normoxia for 7 days followed by the injection of 20 mg/kg SU5416 at day 8 and hypoxia from day 8 to day 21; SuHx-3 w, the rats were injected with 20 mg/kg SU5416 at day 0 and exposed to hypoxia from day 0 to day 21. Rats in the hypoxic chamber received 24 hours of continuous hypoxic exposure. Subsequentially, all the rats were sacrificed on day 21 of the experiment for hemodynamic assessment and tissue harvesting.

Injection of miRNA153agomir in vivo

The effect of miRNA153agomir on PH was detected by injection of miRNA153agomir through the tail vein. Adult male SD rats (n = 40) were randomly divided into the following groups:normoxia group (control), hypoxia group (SuHx), miRNA-NC control group (miR-NC), and miRNA153 overexpression group (miR153agomir) (Figure 1). The rats in normoxia group were housed under normoxia (21% O2) from 0 to 21 days; rats in the SuHx group were injected with SU5416 at 0 day and then maintained in a hypoxia chamber (10% O2) from day 0 to day 21; For miRNA-NC and miRNA153agomir groups, miR-NC (40 nmol) and miR153agomir (40 nmol) were given to rats via tail vein injection (0.3 ml) at day 8, 11, 14 and 17 after treatment with SU5416 plus hypoxia at day 0, respectively. The miR153agomir and miR-NC were designed by GenePharm (Shanghai, China). At 21 days, all the rats were sacrificed for next investigation. The researchers who measured the outcomes did not know from the beginning which group the rats belonged to mir-NC or mir153agomir group.

Figure 1. Study protocol. The diagram illustrates the grouping of experimental animals.

Assessment of the average right ventricular systolic pressure (RVSP)

Before measuring the pressure, all rats were anesthetized with an intraperitoneal injection of pentobarbital (40 mg/kg). According to previous studies (24), the value of RVSP, an average of right ventricular peak systolic pressures was very close to the pressure of the pulmonary artery; hence, RVSP was measured. After the right external jugular vein was successfully isolated, a microcatheter connected with a pressure transducer was gently inserted approximately 4 cm along the direction of the vein into the right ventricle. Blood pumping into the catheter was observed as it reached the correct position. RVSP was recorded continuously for 2 min after the blood pressure had stabilized. Data were collected and analyzed by the BL-420S bioelectrophysiological recorder system (Taimeng, Chengdu, China), which calculated the mean value according to the peak and trough.

Right ventricular hypertrophy and pulmonary histomorphology studies

Following RVSP measurement, all the rats were then euthanasized with an overdose of pentobarbital (100 mg/kg). After cessation of breathing and heartbeat, lung tissues, pulmonary arterial tissues and heart samples were isolated, and then weighed. The right ventricle (RV) was dissected from the left ventricle and septum. Then, the index of right ventricular hypertrophy, RV/(LV+S) ratio was calculated. Right lobes from lung tissues were fixed with 4% paraformaldehyde, dehydrated, paraffin embedded, and sliced into 5 μm sections. Hematoxylin and eosin staining was performed following the histopathological procedures. MT%, which was calculated as the media thickness/vascular outer diameter, was used as the index to measure pulmonary vascular remodeling. Media thickness = vessel circumference (within the media) /2π- vessel lumen circumference (within the intima) /2π, MT% = (media thickness ×2)/(vessel circumference /π) ×100%. Three measurements were performed for each rat, and at least 15 vessels were measured in a blinded manner at 400× magnification (Olympus, Japan). All slides were observed under a light microscope and quantitatively analyzed by Image-Pro Plus software.

Cell culture and treatment

Rat PASMCs were purchased from Jiniou (Guangzhou, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ humidified incubator. In vitro, exposure to hypoxia was achieved in an incubator chamber (Huaxi, Changsha, China) gassed with 3%, 5% CO₂, and 94% N₂.

The miR153mimic and miR-NC were designed by GenePharm (Shanghai, China) and transfected into PASMCs using GP-siRNA-Mate plus according to the instructions. Cyclosporine (CsA) was diluted with saline solution (0.03 mg/ml) for immediate use.

Cell viability assay

The cells (5x103 cells/well) were seeded in 96-well plates and treated with either miRNA153mimic or miRNA-NC before exposure to hypoxia. After hypoxic exposure for 48 h, CCK-8 solution was added according to the manufacturer’s protocol. Finally, the proliferation rates of PASMCs were calculated by measuring their excitation at 450 nm.

Quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNAs were isolated from pulmonary arterial tissues, and PASMCs were isolated using Trizol (Invitrogen, USA) according to the manufacturer’s instructions. The concentration and quality of RNA were measured by an ultraviolet spectrophotometer. According to the instructions (RiboBio Co.; Guangzhou, China), a 10 µl reverse transcription reaction sample addition system was prepared, followed by amplification and quantification using the SYBR-labeled dye method. The primers of miRNA153-3p, NFATc3, GAPDH, and U6 were synthesized by RiboBio (Guangzhou, China). The reverse transcription procedure for U6 small nuclear RNA and miRNA-153 was: 42°C for 60 min, 72°C for 10 min, and storage at 4°C. The reverse transcription procedure for GAPDH, and NFATc3 was: 42°C for 45 min, 85°C for 5 min and storage at 4°C. The PCR reaction program was as follows: Pre-denaturation at 95°C for 10 min, followed by 40 cycles of 95°C denaturation for 10 sec and annealing at 62°C for 20 sec and 70 °C for 10 sec. The relative expression level was quantified with the 2^−∆∆Cq method.

Immunofluorescence

For observing nuclear translocation of NFATc3, sections of lung tissues and cells grown on confocal dishes (Beyotime, Inc; Shanghai, China) were prepared. According to the instructions, paraffin sections were hydrated and then placed into sodium citrate solution (ServiceBio; Wuhan, China) for antigen repair. PASMCs were fixed with the neutral cell fixation solution for 20 min at room temperature, and then they were permeabilized with 0.3% TritonX-100 for 30 min. Tissue sections and cell slides were blocked with goat serum for 30 min at room temperature, followed by incubation with the primary antibody NFATc3 (cat.sc8450; 1:100; Santa Cruz Biotechnology Inc; Texas, USA) at 4°C overnight, after which CY3-conjugated (cat.BA1031; 1:300; Boster, Inc; Wuhan, China) or FITC-conjugated (cat.BA1101; 1:50; BOSTER, Inc; Wuhan, China) secondary antibody was added for 1 h at 37°C. After the addition of the IgG secondary antibody, all operations were carried out in a dark place. Next, antifade mounting medium with DAPI (cat.P0131 Beyotime, Inc; shanghai, China) was used to stain the nuclei for 3 min, before the cover glass slide was mounted. Finally, the images were captured with fluorescence microscope (Olympus).

Flow cytometry

Adherent PASMCs were digested with EDTA-free trypsin, centrifuged at 300 xg for 5 min at 4°C and washed three times with pre-cooled PBS. Subsequently, 400 µl Annexin V (BestBio; Guangzhou, China) binding buffer was added for suspension of the cells (1x10⁶ cells/ml). Afterward, the cells were treated with 5 µl AnnexinV-PE and 8 µl 7-AAD, which stained the nuclei, and incubated under dark conditions for 15 min at 4°C. The stained samples were maintained on the ice until detection by a flow cytometer (BD Biosciences; San Jose, USA). Data were analyzed by FlowJo software.

Western blot

Total protein extraction reagent (cat.BB-3101; BestBio; Guangzhou, China) and nuclear protein extraction reagent (cat.BB-3102; BestBio; Guangzhou, China) were used to isolate total and nuclear proteins from homogenized lung pulmonary arterial tissue and PASMCs. Bicinchoninic acid (BCA) protein assay kit (cat.P0010S; Beyotime, Inc; Shanghai, China) was used to detect the protein concentration in each sample. Loading buffer (cat.P0015L; Beyotime, Inc; Shanghai, China) and PBS were added to maintain an equal concentration of protein (3 µg /µl) in each group. The protein (30 µg) was loaded into 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then immobilized on a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 2 h at room temperature and washed three times using 0.1% Tris-buffered saline with Tween 20 (TBST) for 5 min each time. Then, the PVDF membrane was incubated with primary antibodies against PCNA (cat.A00125; 1:300; Boster, Inc; Wuhan, China), β-actin (cat.Ab8226; 1:1000; Abcam; USA), Kv1.5 (cat.PB0693; 1:1000; Boster, Inc; Wuhan, China), NFATc3 (cat.sc8450; 1:700; Santa Cruz Biotechnology Inc; Texas, USA), Bax (cat.14796S; 1:1000; Cell Signaling technology; USA), Bcl-2 (cat.BA0412; 1:1000; Boster, Inc; Wuhan, China), and LainB1 (cat.ab16048; 1:1000; Abcam; USA) overnight at 4°C; and with goat anti-rabbit IgG (cat.ab6721; 1:10000; Abcam; USA) or rabbit anti-mouse IgG (cat.ab6728; 1:5000; Abcam; USA) secondary antibodies at room temperature for 2 h. After that, the bands were washed with TBST for 10 min and visualized using a sensitive chemiluminescence kit (cat.BL520A; Biosharp Life sciences; Anhui, China) under dark conditions. The relative expression of experimental proteins was quantified and analyzed with ImageJ software.

Statistical analysis

According to recent research (25), all data were expressed as mean ± standard error of mean (SEM). GraphPad Prism 8.0.1 software was employed for measurement analysis. The unpaired Student t-test was used for analysis of two groups, while differences among groups were analyzed by one-way analysis of variance (ANOVA) followed by a Dunnett’s or Bonferroni’s post-hoc test. P value<.05 was considered to indicate a statistically significant difference after adjustment.

Results

SuHx successfully induces a PH model and downregulates miRNA153 expression in the pulmonary arterial tissue

To assess whether a rat model of pulmonary hypertension was successfully established, the right ventricular systolic pressure (RVSP), the ratio of the right ventricular mass to the sum of the left ventricular and septal masses (RV/(LV+S)), and media thickness (MT%) were measured. As shown in Figure 2a, the RVSP was increased significantly in a time-dependent manner (p < .01). SuHx-3 w had significantly higher RVSP value than did the SuHx-1 w and SuHx-2 w (SuHx-3 w vs SuHx-2 w: p < .01; SuHx-3 w vs SuHx-1 w: p < .01). Compared with the N group, the RV/(LV+S) ratio in the SuHx-3 w group was increased from 0.23 ± 0.02 to 0.39 ± 0.01 (Figure 2b). In hypoxia-exposed groups, the RV/(LV+S) ratio had no increase in SuHx-2 w when compared with SuHx-3 w (p > .05). H&E staining (70–100 μm, 400× magnification) demonstrated that the medial wall thickness of pulmonary arterioles (MT%) number was significantly increased in the group of rats treated with SuHx-3 w in contrast to the normal group (Figure 2c, d p < .01). It was observed that the MT% value of SuHx-3 w was significantly higher than that of SuHx-2 w (p < .01) or SuHx-1 w (p < .01). To determine the expression of miRNA153 in the pathogenesis of SuHx-induced PH, we performed RT-qPCR to analyze the miRNA153 relative expression isolated from the pulmonary artery of rats that were exposed to SuHx or normoxia (Figure 2f). Results of RT-qPCR analysis showed a significant decrease in miRNA153 expression in rats from the SuHx-3 w group in contrast to the N group (p < .01). Similarly, compared with SuHx-2 w and SuHx-1 w, the level of miRNA153 from SuHx-3 w was decreased (p < .01 and p < .01, respectively).

Figure 2. SuHx successfully induces PH model and downregulates miRNA153 expression in pulmonary artery tissue. (a) RVSP was measured by right-sided heart catheterization with a pressure transducer microcatheter. (b) RV was dissected from the left ventricle and septum then weighted RV/(LV+S) ratio of each group. (c) The medial thickness percentage (MT%) of rat pulmonary arteries in normal and SuHx groups. (d) Representative images of hematoxylin-eosin (h&e) staining (70–100 μm, 400× magnification). (e, g) Western blot analysis of NFATc3 total protein and nucleoprotein in pulmonary artery tissues. (f, h) RT-qPCR analysis of miRNA153 and NFATc3 expression in the pulmonary artery tissue. N = 10 for each group. Comparisons were performed using one-way ANOVA followed by Bonferroni’s post hoc test. *P < .05, **P < .01 vs normal group (n); ^#P < .05, ^##P < .01 vs SuHx-3 w group. The results are expressed as the mean ± SEM. SuHx, SU5416 combined with hypoxia; PH, pulmonary hypertension; miRNA153, microRNA153; RVSP, right ventricular systolic pressure; RV, right ventricle; LV, left ventricle; S, septum; H&E, hematoxylin and eosin RT-qPCR, quantitative real-time polymerase chain reaction; SEM, standard error of mean.

Together, these results revealed that SU5416 combined with hypoxia for 3 weeks successfully established the animal model of PH and right heart dysfunction as well as marked changes in miRNA153 expression.

Expression level of NFATc3 in rats with PH

In order to explore whether NFATc3 was regulated, RT-qPCR and western blot assays were performed to evaluate the changes in the mRNA and protein expression levels. The relative expression of NFATc3 mRNA was identified to be about 3-fold changes in the SuHx-3 w group compared with the control group (p < .01, Figure 2h). Considering that nuclear translocation is a form of NFATc3 activation, we extracted and quantified nuclear proteins and total proteins from pulmonary arterial tissues. The results of western blot showed that the relative values of the total protein and nuclear fraction in rats treated with SuHx were higher than those in rats under normoxia (p < .01, Figure 2e, g). Notably, the relative level of nucleoprotein in the SuHx-3 w group was significantly increased when compared with the N group (p < .01). Collectively, these results, especially increased NFATc3 unclear translocation in SuHx-induced PH, may contribute to the formation of pulmonary vascular remodeling in PH.

MiRNA153 alleviates SuHx-induced PH and right ventricular function in rats

To investigate the role of miRNA153 in the development of SuHx-induced PH, we injected miRNA153agomir through the tail vein (40 nmol). SU5416 combination with hypoxia for 3 weeks significantly increased the RVSP compared to that in the control group, whereas RVSP was decreased by 25% after overexpression of miRNA153 (p < .01, Figure 3a, b). RV/(LV+S) ratio, a marker of right ventricular hypertrophy, decreased from 0.39 ± 0.02 to 0.33 ± 0.01 after treatment with miRNA153agomir (p < .05, Figure 3c). To detect the effects of miRNA153 on pulmonary arterial remodeling, MT% of the small pulmonary arteries by H&E was performed. The results showed significant vascular occlusion and muscularization in the distal pulmonary arteries in the SuHx group compared to the control group. Moreover, MT% was significantly reduced in miRNA153agomir-treated rats compared with the vehicle-treated groups (p < .05, Figure 3d, e). These data suggested that miRNA153 treatment can reduce the hemodynamic index and prevent the muscularization of small pulmonary arteries observed in SuHx-induced PH.

Figure 3. MiRNA153 alleviates SuHx-induced PH and right ventricular function in rats. (a) RV systolic pressure traces. (b) RVSP was measured by right-sided heart catheterization with a pressure transducer microcatheter. (c) RV/(LV+S) ratio. (d)The media wall thickness of small pulmonary artery. (e)Representative images of H&E staining of lung sections (70–100 μm, 400× magnification). N = 10 for each group, *P < .05, **P < .01 vs control group; ^#P < .05, ^##P < .01 vs miR-NC group. The results are expressed as the mean ± SEM. miRNA153, microRNA153; SuHx, SU5416 combined with hypoxia; PH, pulmonary hypertension; RVSP, right ventricular systolic pressure; RV, right ventricle; LV, left ventricle; S, septum; H&E, hematoxylin and eosin SEM, standard error of mean.

Effect of miRNA153 on NFATc3 in SuHx-induced PH

Our team has confirmed that NFATc3 is a direct target of miRNA153 in human PASMCs (20). To test the mechanism of miRNA153 inhibition in NFATc3 expression, we treated rats with miRNA153agomir and examined the ensuing effects on SuHx-induced PH. The relative expression of miRNA153 was increased after overexpression miRNA153 (p < .01, Figure 4a). RT-qPCR of the pulmonary arterial tissue from rats showed that the relative mRNA level of NFATc3 was increased in the SuHx group when compared to the control group, and it was inhibited in the miR153agomir group (p < .01, Figure 4b).

Figure 4. Effect of miRNA153 on NFATc3 in SuHx-induced PH. (a, b) RT-qPCR analysis of mRNA153 and NFATc3 mRNA expression in the pulmonary artery tissues of rats after treatment of miR153agomir. (c, d, e) The protein levels of NFATc3 and Kv1.5 in pulmonary artery tissue of rats were detected by western blot assay. (F)The expression of NFATc3 in pulmonary artery tissue was assessed by immunofluorescence staining (100× magnification). N = 10 per group *P < .05, **P < .01 vs control group; ^#P < .05, ^##P < .01 vs miR-NC group. All data are mean ± SEM. miRNA153, microRNA153; NFATc3, nuclear factor of activated T cell isoform c3; SuHx, SU5416 combined with hypoxia; PH, pulmonary hypertension; RT-qPCR, quantitative real-time polymerase chain reaction; Kv1.5, potassium voltage-gated channel subfamily A member 5; SEM, standard error of mean.

To clearly describe the nuclear translocation of NFATc3, immunofluorescence was conducted. As shown in Figure 4f, immunofluorescence staining was significantly upregulated in lung tissue from the SuHx group compared to the control group, which was reversed by over expression of miRNA153. Similarly, the results obtained by western blot analysis demonstrated that the level of total protein of NFATc3 in pulmonary arterial tissue was upregulated in the SuHx-PH group compared with the control group, whereas it was decreased in the miR-153agomir group (p < .01, Figure 4c, d,). It was reported that decreased the potassium voltage-gated channel subfamily A member 5 (Kv1.5) inhibited mitochondria-dependent apoptosis (26), which increased the imbalance of proliferation/apoptosis. Consistent with the previous reports, the protein expression of Kv1.5 was reduced in the pulmonary arterial tissue of SuHx-induced PH, and preventive administration of miR153agomir significantly increased the expression level of Kv1.5 (p < .01, Figure 4e). Additionally, miRNA153 stimulated the expression of Kv1.5 via suppressing NFATc3 expression in SuHx-induced PH.

Effect of hypoxia on the expressions of miRNA153 and NFATc3 in PASMCs

To further explore the role of hypoxia in miRNA153 and the level of NFATc3, we examined the different exposure times to hypoxia in PASMCs. Hypoxia treatment led to a significant decrease in the expression of miRNA153 and an increase in the level of NFATc3 mRNA in PASMCs, which was analyzed by RT-qPCR (p < .01, Figure 5a, b). As shown in Figure (5c, d), hypoxia promoted the expression levels of total protein and nucleoprotein of NFATc3 in PASMCs. Exposure to 48 hours of hypoxia resulted in a significant increase in NFATc3 total protein and nucleoprotein levels as compared with the hypoxia-0 h group (p < .01). The total protein and nucleoprotein levels of NFATC3 in PASMCs cultured with normal oxygen for 48 hours did not change significantly as compared with those cultured for 0 hour (P > .05). Immunofluorescence assays were conducted to characterize the localization of NFATc3 in the cytoplasm and nucleus in detail (Figure 5e). Hypoxia treatment (48 h) significantly stimulated nuclear translocation, which was indicated by strong nuclear staining. These results support the claim that NFATc3 was up-regulated and miRNA153 was down-regulated in PASMCs under hypoxic conditions.

Figure 5. Effect of hypoxia on the expressions of miRNA153 and NFATc3 in PASMCs. (a, b) The mRNA expression of miRNA153 and NFATc3 in PASMCs determined by RT-qPCR. (c) Western blot analysis of NFATc3 total protein in PASMCs. (d) The protein from nucleus of PASMCs analyzed with western blotting. (E)Immunofluorescence images showing translocation of NFATc3 (green) into DAPI-stained nuclei (blue) in normoxia and hypoxia treated PASMCs (400× magnification). Comparisons were performed using one-way ANOVA followed by Dunnett’s post hoc test. *P < .05, **P < .01 vs hypoxia 0 h group. The results are represented as the mean ± SEM. N, normoxi Hy, hypoxia; miRNA153, microRNA153; PASMCs, pulmonary arterial smooth muscle cells; RT-qPCR, quantitative real-time polymerase chain reaction; NFATc3, nuclear factor of activated T cell isoform c3; SEM, standard error of mean.

Effect of miRNA153 on hypoxia-induced proliferation and resistance to apoptosis of PASMCs

Hypoxia decreased the expression of miRNA153 as compared to normoxia in PASMCs, which was increased after transfection with miR153mimic (p < .01, Figure 6a). As the effect of miRNA153 has been revealed in vivo, we next sought to establish whether miRNA153 inhibits proliferation and induces apoptosis of PASMCs. First, we detected the effects of miRNA153 on hypoxia-induced cell proliferation in PASMCs by western blot assay and CCK-8 assays. As illustrated in Figure 6 b, c, e, the level of cell proliferation was promoted by hypoxia and reversed by overexpression miRNA153 (p < .01). Second, we detected the protein of Bax and Bcl-2, which are critical to apoptosis. As demonstrated by western blotting, there was a significant down-regulation of Bax/Bcl-2 ratio in hypoxia-induced PASMCs compared with normoxia, which was reversed by miR153 overexpression (p < .01, Figure 6c, d). Third, to determine whether overexpression of miRNA153 induced apoptosis of PASMCs exposed to hypoxia, flow cytometry was performed. As shown in Figure 6f, hypoxia profoundly decreased the apoptosis and treatment with miRNA153 increased the apoptosis of PASMCs (p < .01). These data demonstrated that miRNA153 effectively augmented the anti-proliferation and pro-apoptotic effects in PASMCs upon hypoxia exposure.

Figure 6. Effect of miRNA153 on hypoxia -induced proliferation and resistance to apoptosis of PASMCs. (a) The expression of miRNA153 in PASMCs after transfection with miR153mimic assessed by RT-qPCR. (b) The viability of PASMCs obtained by CCK-8. (c, d, e) Immunoblot analysis of PCNA, Bax, Bcl-2 from PASMCs. (f) Using flow cytometer for apoptosis to assess miR153mimic on hypoxia-induced PASMCs. *P < .05, **P < .01 vs normoxia group; ^#P < .05, ^##P < .01 vs miR-NC group. The results are expressed as the mean ± SEM. miRNA153, microRNA153; PASMCs, pulmonary arterial smooth muscle cells; SEM, standard error of mean.

CsA increases the expression of Kv1.5 in PASMCs after hypoxia exposure

Given that hypoxia increases the expression of NFATc3, which plays a critical role in the development of PH and vascular remodeling in mice (27). In addition, previous studies have shown that NFAT activation by hypoxia decreases Kv1.5 expression in PASMCs (28). Therefore, we tested the effects of CsA, a direct inhibitor of NFAT, on Kv1.5 protein in vitro after hypoxia exposure. Following 3% O₂ exposure, the protein expression level of NFATc3 was increased (p < .01, Figure 7a, b) and the level of Kv1.5 was reduced (p < .01, Figure 7a, C) in PASMCs compared with the normoxia group, which were subsequently reversed following CsA treatment.

Figure 7. CsA increase the expression of Kv1.5 in PASMC after hypoxia exposure. (a) The protein trace of NFATc3 and Kv1.5 in PASMCs were detected by western blot assay. (b, c) Western blot analysis of NFATc3 and Kv1.5 in PASMCs treated with CsA. Comparisons were performed using one-way ANOVA followed by Bonferroni’s post hoc test. *P < .05, **P < .01 vs normoxia group; ^#P < .05, ^##P < .01 vs Hy-48 h group. The results are expressed as the mean ± SEM. CsA, Cyclosporine; Kv1.5, potassium voltage-gated channel subfamily A member 5; PASMCs, pulmonary arterial smooth muscle cells; SEM, standard error of mean.

Overexpression of miRNA153 reverses the hypoxia-reduced expression of Kv1.5 in PASMCs

To determine the role of miRNA153, we examined the protein level of Kv1.5 in hypoxic PASMCs treated with miRNA153mimic, by western blot and immunofluorescence. After having demonstrated that miR153 targeted sites within the 3′UTR of NFATc3mRNA and inhibited the expression in vivo, we next evaluated the consequences of in vitro miRNA153 inhibition on NFATc3 at the protein level. Immunofluorescence staining of PASMCs from rats confirmed that nuclear fraction of NFATc3 was strongly increased in the miR-NC group and restored to a level similar to normoxia in the miR153mimic group (Figure 8a). As shown in Figures 8 b and c, there was an apparent increase in NFATc3 expression in hypoxic PASMCs, and when miRNA153 was overexpressed, the hypoxia-induced increase was evidently reversed (p < .01). The expression of Kv1.5 was downregulated in PASMCs exposed to hypoxia compared with the normoxia group (p < .01, Figure 8b, d). However, the level was higher in the miRNA153mimic group than in the miR-NC group, as determined by western blot (p < .01). In addition, overexpression of miRNA153 significantly decreased the activity of NFATc3 and protein expression, and it increased the expression of Kv1.5 in the PASMCs of rats with hypoxia treatment.

Figure 8. Over expression of miRNA153 reverses the hypoxia-reduced expression of Kv1.5 in PASMCs. (a) Immunofluorescence images showing translocation of NFATc3(red) into DAPI-stained nuclei (blue) in PASMCs (400× magnification). (b, c, d) Immunoblot analysis of NFATc3 and Kv1.5 from PASMCs treated with miR153mimic. *P < .05, **P < .01 vs normoxia group; ^#P < .05, ^##P < .01 vs miR-NC group. The results are expressed as the mean ± SEM. miRNA153, microRNA153; Kv1.5, potassium voltage-gated channel subfamily A member 5; PASMCs, pulmonary arterial smooth muscle cells; NFATc3, nuclear factor of activated T cell isoform c3; SEM, standard error of mean.

Discussion

PH is a malignant vascular disease that is caused by abnormally elevated pulmonary arterial pressure and is characterized by hypertrophic hyperplasia, decreased elasticity, and reduced narrowing of the pulmonary vessels (29). PASMCs are an important part of the vascular muscle, which is involved in phenotypic maintenance and contraction (30,31). Hypoxia induces an imbalance in proliferation/apoptosis in PASMCs that leads to irreversible changes in pulmonary vessels (32,33), which further aggravate the progression of the disease. SU5416, a vascular endothelial growth factor receptor antagonist and the combination of chronic hypoxia/SU5416 produces an animal model sharing many hallmarks of human pulmonary arterial hypertension including severe vascular remodeling, endothelial dysfunction, and plexiform lesions, those are not present with chronic hypoxia alone (34,35). Accordingly, in this study, SU5416 was used in combination with hypoxia for 3 weeks to establish a rat model of PH, as reported in reference (36). It was found that RVSP, RV/(LV+S), MT%, remodeling of the medium and small vessels were significantly higher in the hypoxia/SU5416 treated rats. These results indicated that the animal model of PH was successfully established in the present study, which may provide a good and convenient model for the future investigation of PH.

NFATc3, the most influential member of the NFAT family, might be an ideal target for PH because of its selectivity and effectiveness in PH (9,37) .Chronic hypoxia increased endothelin 1 and intracellular Ca²⁺ concentrations in PASMCs, leading to calcineurin activation and promotion of NFAT transcription and activation (38). In addition, the translocation of dephosphorylated NFAT from the cytoplasm to the nucleus is essential for the subsequent activation of its target genes (39). In the present study, the authors further verified these results in SuHx rats. After the exposure of hypoxic conditions to SuHx rats from 0 to 21 days, the total protein and nucleoprotein levels of NFATc3 were dramatically upregulated in the pulmonary arterial tissues. At the same time, the RNA expression of NFATc3 was increased from 1 week after exposure to hypoxia, which proved hypoxia promoted the activation of NFATc3, and the translocation from the cytoplasm to the nucleus was induced by SuHx.

Accumulating research has confirmed that miRNAs, which are active biological regulatory factors, are involved in the proliferation, migration, and apoptosis of PASMCs by regulating gene function, and it has documented key roles in the vascular remodeling of PH (40,41). The team of authors from the present study has demonstrated that miRNA153 directly binded to the 3′-UTR of NFATc3 and reversed the hypoxia induced overexpression of NFATc3 and the excessive proliferation and migration of PASMCs (20). The SuHx rat PH model was established to investigate the effect of miRNA 153 on pulmonary hypertension in animals. It was found that the miRNA153 expression level was significantly downregulated in response to SuHx in 3 weeks. After miRNA153 agomir was injected into the tail vein of the SuHx-induced PH rats, miRNA153 was observed to be highly expressed in pulmonary arterial tissues, which proved the animal model of the miRNA153agomir group was successfully established. It was also discovered that treatment with miRNA153agomir markedly decreased medial wall thickening and reversed the remodeling of the vessel wall in vivo, and miRNA153mimic could effectively reverse hypoxia-induced proliferation and apoptosis of PASMCs. The role of miRNAs in the development of PAH as well as on their potential use as biomarkers and therapeutic tools in both experimental PAH models and in humans has been investigated (42).This study identifies miRNA153 as a potential target for the treatment of PH, and miRNA153 agomir may be a potential new therapy to mitigate PH.

Kv1.5 is the downstream factor of the NFAT family. Kang et al. reported that Di-chloroacetate (DCA) inhibits chronic hypoxia-induced PAH by inhibiting NFATc2 and increasing Voltage-gated potassium (Kv) 1.5 (43). Sebastien et al. reported the inhibition of NFAT increased the level of Kv1.5 (44). The effect of miRNA153 on NFATc3 and Kv in cell experiments was initially explored in the present study. Following 3% O2 exposure, the protein expression level of NFATc3 was increased and the level of Kv1.5 was reduced in PASMCs compared with the normoxia group, which were subsequently reversed following cyclosporine (CsA, a NFAT3 inhibitor) treatment. Similar results also appeared after transfection with miRNA153mimic in PASMCs. Further studies are required to explore whether there is a direct link between miRNA153 and Kv1.5. Despite the CsA targets NFAT and may reverse PAH in animals (44,45), one limitation of our study involving the CsA was used only in PASMCs. In the future, NFAT3 inhibitor will be used in animal model to explore the role of NFATc3 in PAH. Moreover, other members including NFATc1, NFATc2, NFATc4, and NFATc5 also play a critical role in the pathogenesis of PH (22) .Thus, the subsequent research about NFAT family is indubitably required to apply.

Conclusion

This paper demonstrated that forced overexpression of miRNA153 alleviated the pulmonary arterial blood flow resistance and vascular remodeling in SuHx-induced PH in vivo and increased apoptosis functions of PASMCs in vitro via inactivation of the NFATc3/Kv1.5 pathway. In summary, miRNA153 plays an important role in the pathogenesis of protection against SuHx-induced PH, and it provides a theoretical basis for further study of PH.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The present study was supported by Liaoning Revitalization Talents Program (No. XLYC2007142) and Doctoral Fund of Liaoning Province (No.2019-BS-100).