Chronic hypoxia is one of the most common causes of secondary pulmonary hypertension, the mechanisms of which remain unclear. MicroRNAs (miRNAs) are small, noncoding RNAs that inhibit the translation or accelerate the degradation of mRNA. Previous studies have demonstrated that deregulated miRNA expression contributes to various cellular processes including cell apoptosis and proliferation, which are mediated by hypoxia. In the present study, the expression of miR-98 was identified to be decreased in the lung tissue of a hypoxic pulmonary hypertension (HPH) rat model and pulmonary artery (PA) smooth muscle cells (PASMCs), which was induced by hypoxia. By transfecting miR-98 mimics into PASMCs, the high expression of miR-98 inhibited cell proliferation, but upregulated hypoxia-induced PASMCs apoptosis. However, these effects of miR-98 mimics on PASMCs were reversed by ALK1 (activin receptor-like kinase-1) overexpression. ALK1 was identified as a candidate target of miR-98. In addition, overexpressing miR-98 markedly decreased the pulmonary artery wall thickness and the right ventricular systolic pressure in rats induced by hypoxia. These results provided clear evidence that miR-98 was a direct regulator of ALK1, and that the downregulation of miR-98 contributed to the pathogenesis of HPH. These results provide a novel potential therapeutic strategy for the treatment of HPH.
Pulmonary hypertension (PH) is a physiological pathological disorder with several clinical manifestations, which finally leads to cardiovascular and respiratory diseases (
MicroRNAs (miRNAs) are small endogenous noncoding RNAs and they serve an important role in regulating gene expression by targeting the 3′-untranslated region (3′-UTR) of mRNA to repress its translation or accelerate its degradation (
Transforming growth factor-β1 (TGF-β1), which is a multifunctional cytokine, serves an important role in regulating cell differentiation, proliferation and extracellular matrix deposition, which is directly associated with the occurrence and development of pulmonary hypertension (
In the present study, the role of miR-98 in HPH was examined. It was identified that the levels of miR-98 expression were decreased in the lung tissues of HPH rat models and rat PASMCs under hypoxia, as compared with those of the controls. Furthermore, the downregulation of miR-98 was involved in the hypoxia-induced proliferation and apoptosis of PASMCs. In addition, it was demonstrated that ALK1 was the direct target of miR-98. The conclusions of the present study provided novel insights into the pathogenesis of HPH and potential targets for its clinical diagnosis and treatment.
Adult male Wistar rats (weighing 200±10 g; age, ~8 weeks) were obtained from the Animal Experimental Centre of Nanjing Medical University (Nanjing, China). A total of ~60 rats were used in the experimental model, and were fed with standard rat chow and allowed water
PA segments were collected from a group of 5 rats not included in the HPH rat model immediately following sacrifice. The samples were then minced into small fragments using ophthalmic scissors, followed by digesting with 0.25% trypsin (Thermo Fisher Scientific, Inc.) at 37°C for 40 min. Next, 0.2% collagenase (Merck KGaA) was added to treat the fragments at 37°C for an additional 4 h. Cell suspension was filtered and dissociated with a 40 µm cell strainer (BD Biosciences), followed by centrifugation at 850 × g for 10 min at 4°C. Rat PASMCs were incubated in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) supplied with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified incubator. The cell line 293 was obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science and maintained in DMEM with 10% FBS at 37°C in a humidified incubator.
Genes targeted by miR-98 were predicted using PicTar (version 2;
PAMSCs at ~60% confluence were exposed to difference oxygen concentrations (0, 3 and 5%) by connecting the incubator with a chamber that was equilibrated with a water-saturated gas mixture of (0, 3 and 5%) O2, 5% CO2 and (95, 92 and 90%) N2 at 37°C for 24–48 h, as described previously (
A total of 24 h prior to transfection, suspended rat PASMCs at 50% confluence were seeded onto 12-well plates. The miR-98 mimics (5′-UGAGGUAGUAAGUUGUAUUGUU-3′), inhibitors (5′-AACAAUACAACUUACUACCUC-3′) or negative control (NC) sequence, (5′-GUGUAACACGUCUAUACGCCCA-3) were purchased from Guangzhou RiboBio Co., Ltd. Mimics or inhibitors were transfected into PASMCs with Lipofectamine® 3000 reagent (Thermo Fisher Scientific, Inc.). The final concentration of miRNAs was 60 nM. To overexpress ALK1 in rat primary PASMCs, coding sequences of ALK1 were cloned into a lentiviral vector (GeneCopoeia, Inc.) (lenti-ALK1) and packaged into lentiviruses particles by Obio Technology (Shanghai) Co., Ltd. The transfected cells (60% confluence, 6 h after transfection) were incubated with lentiviruses for ~6 h at 37°C, then the media was replaced with normal media. After 24, 48 or 72 h, the cells were harvested for subsequent experiments.
TRIzol® reagent (Thermo Fisher Scientific, Inc.) was used to extract total RNA from lung tissues or PASMCs according to the manufacturer's protocol. RNAs (1 µg) was used for cDNA reverse transcription using an M-MLV kit (Takara Biotechnology Co., Ltd.). RT-qPCR was performed for the detection of ALK1 and ALK4 gene expression. The forward and reverse primers were as follows: Rat ALK1 forward, 5′-ACCCAAACTCCTTCGGAGGAG-3′; rat ALK1 reverse, 5′-CGCTGCTTCTCCTGCCTTC-3′; rat ALK4 forward, 5′-TGACCTGAGGGTGCCCAGTG-3′; rat ALK4 reverse, 5′-TGAGGGGTCCTCCATGTCCAG-3′; β-actin forward, 5′-GAGTACGATGAGTCCGGCCCC-3′; and β-actin reverse, 5′-GCAGCTCAGTAACAGTCCGCCT-3′. β-actin was used as an internal control. The relative expression levels of ALK1 and ALK4 were quantified using the Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.) with Power SYBR1 Green PCR Master Mix (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. The relative mRNA expression levels were analyzed through using the expressed relative to the threshold cycle values (ΔCq), and then converted to fold changes using the 2−ΔΔCq method (
The expression levels of miR-98 were analyzed and quantified separately using a stem-loop RT-PCR assay, as described previously (
A Cell Counting Kit-8 (CCK-8; Nanjing KeyGen Biotech, Co., Ltd.) assay was used to analyze cell proliferation, as described previously (
Apoptosis was analyzed using a flow cytometer (BD Biosciences). Briefly, an annexin-V fluorescein isothiocyanate and propidium iodide double-stain assay was performed in accordance with the manufacturer's protocol (FITC Annexin V Apoptosis Detection Kit I; BD Biosciences). The results were analyzed using FlowJo software (version 7.5.5; Tree Star, Inc.). Each experiment was performed in triplicate.
A total of 10 rats from each group was used to measure RVSP by right heart catheterization, as previously described (
The 3′-UTR-Luc reporter of ALK1 was constructed by the ligation of ALK1 3′-UTR PCR product into the
For western blot analysis, total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) and quantified using the Bradford method. A total of ~30 µg total protein from each sample was loaded and separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (EMD Millipore). Following blocking with 5% non-fat milk in PBST buffer at 37°C for 1 h, membranes were incubated with the primary antibody at 4°C overnight. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.; cat. no. sc-2004; 1:2,000 dilution) for 1 h at 37°C and finally detected using an ECL substrate kit (Tanon Science and Technology Co., Ltd.). Chemiluminescence was detected using the ChemiDoc XRS+ (Bio-Rad Laboratories, Inc.). The secondary antibodies conjugated with HRP (cat. no. sc-2004; 1:2,000 dilution) and primary antibodies against β-actin (cat. no. sc-70319; 1:1,000 dilution) were purchased from Santa Cruz Biotechnology, Inc., ALK1 (cat. no. ab108207; 1:1,000 dilution), cleaved caspase 3 (cat. no. ab13847; 1:1,000 dilution), proliferating cell nuclear antigen (PCNA; cat. no. ab92552; 1:1,000 dilution), smad1 (cat. no. ab66737; 1:1,000 dilution) and P-smad1 (cat. no. ab73211; 1:1,000 dilution) primary antibodies were purchased from Abcam. The densitometric was analyzed using ImageJ (version 1.51J8; National Institutes of Health).
After the rats were anesthetized, the lung tissues were obtained immediately, then the tissues were sliced into tissue blocks, and immersed in 4% paraformaldehyde for overnight fixation at 37°C. Fixed tissues were then dehydrated in ascending alcohol series, cleared, and embedded in paraffin wax. The tissues were cut into 4-µm thick sections using a Leica slicer (Leica Microsystems, Inc.) and stained with hematoxylin and eosin (H&E) using a standard method as described previously (
All data are presented as the mean ± standard deviation. GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for statistical analysis. Unpaired two-tailed Student's t-test and one-way ANOVA followed by Tukey-Kramer post-hoc test were used to determine statistical significance. P<0.05 was considered to indicate a statistically significant difference.
The HPH rat model was established by subjecting the rats to a hypoxic assault with 10% O2 for 3 weeks, as described previously (
PASMC proliferation is a well-known mechanism of vascular remodeling in HPH. In the present study, a CCK-8 assay was used to assess the effect of miR-98 on PASMC proliferation. As demonstrated in
Furthermore, to explore the molecular mechanism underlying the modulation of miR-98 in PASMC proliferation, PASMC apoptosis was analyzed by flow cytometry, and the results indicated that the overexpression of miR-98 significantly induced cell apoptosis, whereas transfection with anti-miR-98 inhibitors prevented hypoxia-induced apoptosis (
In order to elucidate the molecular mechanism through which miR-98 affects HPH, TargetScan and MiRanda were used to identify putative binding sites for miR-98. A number of mRNAs may be targets of miR-98, including the ALK gene family, which is associated with the occurrence and progression of HPH (
To additionally examine the role of ALK1 in mediating the function of miR-98, rescue experiments were conducted. PASMCs were transiently transfected with miR-98 mimics and a ALK1 overexpression vector carried by lentiviral particles (lenti-ALK1), and subsequently exposed to hypoxia for 24 h or 72 h. Cell survival, proliferation and apoptosis assays indicated that the re-introduction of ALK1 into the miR-98-overexpressing cells inhibited PASMC apoptosis (
To examine the effect of miR-98 agomir, a chemically-modified miR-98 mimic, on the expression of ALK1 and the development of HPH, an animal model of HPH was established by exposing rats to hypoxia and a tail vein injection of miR-98 agomir or its control. The rats were divided into three groups, including normoxia control (N-control), hypoxia control (H-control) and hypoxia + miR-98 agomir (H + miR-98 agomir). The results demonstrated that the miR-98 agomir injection significantly increased miR-98 in the lung tissue, as compared with the untreated group. In addition, the mRNA level of ALK1 was significantly decreased following the tail injection of miR-98 mimics (
Although the mechanisms of PH have been studied for several decades, they remain unclear. Chronic hypoxia involving multiple molecular signaling pathways has been reported to be an important reason for the occurrence and development of PH. In previous decades, several studies have demonstrated that the association between miRNAs and vascular remodeling, the development of hypertrophy, failure in the heart muscle function and vasculature, all are relevant (
The present study revealed that the expression of miR-98 was significantly decreased in the lung tissues of the HPH rat model and PASMCs under hypoxia compared with the controls. However, the association between miR-98 and the pathological process of HPH was not examined. Based on all of these results, it was reasonable to hypothesize that miR-98 may serve an important role in the development of HPH. In order to examine our hypothesis, an HPH animal model and primary rat PASMCs were used to examine the function of miR-98 in the development of HPH. As expected, miR-98 overexpression inhibited hypoxia-induced PASMC proliferation. Rat PASMC apoptosis was also markedly increased following miR-98 transfection. These results suggested that miR-98 may serve an important role in hypoxia-induced PASMC hyperproliferation. miRNAs exert their functions via targeting different target genes; for example miR-328 regulates HPH by targeting insulin growth factor 1 receptor and l-Type calcium channel-α1C (
Though gain-of-function approaches, it was confirmed that ALK1 is a direct target gene of miR-98. Firstly, the mRNA expression of ALK1 was identified to be increased in the lung tissues of HPH rats, while that of ALK4, which was revealed to be a target of miR-98 in breast cancer cells, was not (
However, it must be noted that when the PASMCs transfected with miR-98 mimics and ALK1 overexpression lenti-ALK1 particles were exposed to hypoxia for 24 h, and then analyzed ~30 h after transfection, it is possible that the cells had not completely recovered from the toxicity of transfection, and that the levels of overexpression of ALK1 were not high. This may explain why the overexpression of ALK1 did not change the levels of cell apoptosis at first. However, the results from the present study indicated that, after transfection for 72 h, the overexpression of ALK1 significantly inhibited the levels of apoptosis.
In addition, it was indicated that the forced expression of ALK1 was also degraded by overexpression of miR-98; therefore, it may be better to use resistant mutants of ALK1 in future studies. It was concluded that ALK1 is a direct and pivotal target gene of miR-98 in the development of HPH.
ALK1 is a specific type I receptor that transmits signals via the ALK1/Smad1/5 pathways. Several studies have demonstrated that the TGF-β/ALK1 signaling pathway serves a key role in idiopathic pulmonary arterial hypertension and experimental hypoxic PH via a direct effect on pulmonary endothelial cells, leading to the overproduction of growth factors and inflammatory cytokines that are involved in the pathogenesis of PAH (
In conclusion, to the best of our knowledge, the present study was the first to provide evidence that miR-98 serves an important role in vasoconstriction and remodeling in the pathogenesis of HPH. The inhibitory effect of miR-98 on vasoconstriction may be attributable to the hypoxia-induced inhibition of the ALK1 expression. These results provide novel insight into the molecular mechanisms of HPH and suggest a potential target for treatment of HPH. However, these results require verification through additional clinical studies.
Not applicable.
The present study was supported by a grant from The Science and Technology Bureau of Xuzhou (grant no. BK2016109).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
XiaZ conceived and designed the experiments. QL and XinZ performed the experiments, and XiaZ and QL analyzed the data and wrote the manuscript.
All aspects of the present study were approved by the Ethics Committee of the Municipal Hospital Affiliated to Xuzhou Medical University.
Not applicable.
The authors declare that they have no competing interests.
Effect of hypoxia on miR-98 expression
Effect of miR-98 on PASMCs proliferation and apoptosis. Following transfection with miR-98 mimics or inhibitors, the hypoxia-induced PASMC proliferation for (A) 24 or (B) 48 h was analyzed by Cell Counting Kit-8 assay. (C) The protein level of PCNA was determined by western blot analysis following PASMC transfection with miR-98 mimics or inhibitors and subsequent exposure to hypoxia for 24 hz (D) Representative FACS analysis of Annexin V and PI staining. (E) Percentage of apoptotic cells analyzed by FACS. (F) The protein level of cleaved caspase 3 was determined by western blot analysis following PASMC transfection with miR-98 mimics or inhibitors and subsequent exposure to hypoxia for 48 h. (G) Densitometry analysis of the western blots of caspase 3 and cleaved caspase 3. (H) The miR-98 expression in PASMCs transfected with miR-98 mimics or inhibitors was examined by reverse transcription quantitative polymerase chain reaction. *P<0.05 and **P<0.01 (n=3). miR, microRNA; PASMCs, pulmonary artery smooth muscle cells; PCNA, proliferating cell nuclear antigen; PI, propidium iodide; FACS, fluorescence-activated cell sorting; NC, negative control.
ALK1 is a direct target of miR-98 in PASMCs. (A) Diagram of miR-98 seed sequence, indicating that it matched the 3′-UTR of the ALK1 and ALK4 genes. (B) RT-qPCR analysis of the ALK1 and ALK4 expression in lung pulmonary arteries from H and N rats. **P<0.01 (n=10). (C) Luciferase reporter assays in 293 cells, following co-transfection of cells with WT or mut 3′-UTR ALK1 and miR-98 mimics. (D) RT-qPCR analysis of the ALK1 expression 24 h following transfection with miR-98 mimics, inhibitors or NC. (E) Western blot analysis results of the ALK1 expression in PASMCs transfected with miR-98 mimics, inhibitors or NC. (F) Densitometric analysis of the western blot analysis of ALK1. All experiments were repeated 3 times. *P<0.05, **P<0.01. ALK, activin receptor-like kinase; miR, microRNA; UTR, untranslated region; PASMCs, pulmonary artery smooth muscle cells; RT-qPCR; reverse transcription quantitative polymerase chain reaction; N, normoxic; H, hypoxic; NS, not significant; WT, wild type; mut, mutant; NC, negative control.
Confirmation of miR-98 functions by targeting ALK1 in PASMCs. (A) Representative FACS analysis of Annexin V and PI staining of PASMCs following transfection with miR-98 mimics and lenti-ALK1 particles, and subsequent exposure to hypoxia for 24 h. (B) Percentage of apoptotic cells analyzed by FACS. (C) Cell Counting Kit-8 assay was used to analyze PASMC proliferation following transfection of PASMCs and exposure to hypoxia for 72 h. (D) Upper panel, western blot analysis of phosphorylated Smad1 and total Smad1; lower panel, western blot analysis of ALK1, cleaved caspase 3 and PCNA levels in PASMCs transfected with miR-98 mimics and ALK1 overexpression vector and subsequent exposure to hypoxia for 24 h. Densitometric analysis of (E) PCNA and (F) cleaved caspase 3 levels. All experiments were repeated 3 times. **P<0.01. miR, microRNA; ALK1, activin receptor-like kinase-1; PASMCs, pulmonary artery smooth muscle cells; PI, propidium iodide; NS, not significant; CCK-8, Cell Counting Kit-8; lenti-ALK1, ALK1 overexpression vectors carrying lentiviral particles; FACS, fluorescence-activated cell sorting; PCNA, proliferating cell nuclear antigen.
Effect of miR-98 agomir treatment on hypoxia-induced pulmonary vasoconstriction in the HPH rat model. (A) Western blot analysis of ALK1 and phosphorylated Smad1 levels in lung pulmonary arteries from normoxic and hypoxic rats with or without miR-98 agomir treatment. (B) Reverse transcription quantitative polymerase chain reaction analysis of ALK1 and miR-98 expression in lung pulmonary arteries from N and H rats with or without miR-98 agomir treatment. (C) Measurement of RVSP. (D) Representative images of hematoxylin and eosin staining of lung pulmonary arteries (magnification, ×200). (E) Representative images of Masson staining of lung pulmonary arteries (magnification, ×200). (F) Representative immunohistochemical staining of PCNA in N-control, H-control and H + miR-98 agomir treatment groups (magnification, ×400). (G) Semi-quantitative analysis of immunohistochemical staining of PCNA (analyzed by ImageJ). *P<0.05, **P<0.01 and ***P<0.001. All experiments were repeated 3 times. miR, microRNA; HPH, hypoxic pulmonary hypertension; ALK1, activin receptor-like kinase-1; N, normoxia; H, hypoxia; RVSP, right ventricular systolic pressure; PCNA, proliferating cell nuclear antigen; N-control, normoxic control group; H-control, hypoxia induced group; H + miR-98 agomir, hypoxia induced with miR-98 agomir.