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MicroRNAs (miRNAs) have a key role in the pathogenesis of pulmonary arterial hypertension (PAH), a disease characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells. In the present study, miR-760 was demonstrated to be downregulated in PAH lung tissues compared with normal lung tissues, an effect that may be associated with the development of PAH. Hypoxia is an important stimulus for human pulmonary artery smooth muscle cell (hPASMC) proliferation and the occurrence of PAH. Therefore, the effect of miR-760 in hypoxia-treated and normal hPASMCs was investigated. Expression of exogenous miR-760 decreased cell proliferation in hypoxia-induced hPASMCs, and promoted cell apoptosis with an increase in the BCL2 associated X/BCL2 ratio and the expression levels of Caspase-3 and Caspase-9. In addition, overexpression of miR-760 suppressed the migration of hPASMCs under hypoxic conditions. Furthermore, miR-760 was demonstrated to mediate its anti-proliferation effect via the regulation of toll-like receptor 4 (TLR4), a direct target of miR-760. The results revealed that knockdown of TLR4 restrained the hypoxia-induced hPASMC proliferation and induced cell apoptosis. The present study uncovered a novel regulatory pathway involving miR-760 and suggested that miR-760 may be explored as a potential therapy for PAH in the future.
Pulmonary arterial hypertension (PAH), characterized by a progressive increase of lung vascular resistance and pressure, can gradually lead to death (
MicroRNAs (miRNAs) are a kind of endogenous non-coding RNAs found in eukaryotes, with a size of ~20–25 nucleotides, which negatively regulate gene expression by targeting specific messenger RNAs (
miR-760, a biomarker of colorectal cancer, is significantly downregulated in colorectal cancer and correlated with patient clinicopathological features (
Human lung tissues were acquired from idiopathic PAH patients undergoing lung transplantation and from normal controls. This experiment was approved by the Ethics Committee of Nanjing Medical University (Nanjing, China), and informed consent was obtained from all the participants prior to the study.
hPASMCs were isolated from 16 donors not suitable for lung transplantation with PAH, and isolated as previously described (
HPASMCs were cultured in SmBM medium supplemented with 5% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were grown in 5% CO2 at 37°C. For induction of hypoxia, cells were cultured in a hypoxia incubator (Thermo Fisher Scientific, Inc.) with 3% O2, 5% CO2, and balanced nitrogen.
Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) reagent was used to transfect 100 ng of miR-760 mimics (forward, 5′-CGG CUC UGG GUC UGU GGG GA-3′, reverse, 5′-UCC CAC AGA CCC AGA GCC G-3′) and 100 ng of negative control (NC) (forward, 5′-ACG UGA CAC GUU CGG AGA AUU-3′, reverse, 5′-UCC CAC AGA CCC AGA GCC G-3′) for 48 h at 37°C, according to the instructions. Small interfering (si)RNA targeting TLR4 (100 ng) (forward, 5′-GGA CAG CUU AUA ACC UUA ATT-3′, reverse, 5′-UUA AGG UUA UAA GCU GUC CTT-3′) and siRNA control (forward, 5′-UUC UCC GAA CGU GUC ACG UTT-3′, reverse, 5′-ACG UGA CAC GUU CG GAG AATT-3′) were transfected to hPASMCs to inhibit the expression of TLR4 for 48 h at 37°C.
Total RNA was isolated from human lung tissues and hPASMCs with the miRNeasy Mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. cDNA was generated from 2
hPASMCs were confirmed by immunofluorescence staining for α-actin and succinate receptor 1 (SUCNR1, also known as GPR91). Cells were fixed with 4% formaldehyde for 15 min at room temperature and incubated in 5% Tris buffered saline with Tween-20 diluted non-fat dry milk for 1 h. Immunofluorescence staining was performed using α-actin (ab5694; 1:500; Abcam, Cambridge, UK) and GPR91 primary antibody (ab140795; 1:500; Abcam) incubation overnight at 4°C, and Alexa Fluor 488 anti-rabbit IgG secondary antibody (ab150077; 1:1,000; Abcam) incubation at room temperature for 1 h. Cells were incubated using DAPI for nuclear staining at room temperature for 30 min. Microscopic analysis was performed with a confocal laser-scanning microscope (Leica Microsystem GmbH, Wetzlar, Germany). For the EdU assay, EdU was added to the culture medium for 8 h. The cultured cells were then fixed with 4% paraformaldehyde for 20 min. Following incubation with 0.2% Triton X-100 to permeabilize the nuclear membrane for 10 min at room temperature, the cells were blocked with PBS containing 10% goat serum (Gibco; Thermo Fisher Scientific, Inc.) for 1 h at 25°C. Then, hPASMCs were stained using the Cell-Light EdU Apollo 488
Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. The optical density (OD) at 450 nm was recorded on a Microplate Reader (Bio-Rad Laboratories, Inc.).
Apoptosis rates were evaluated by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA) using an Annexin V-FITC Apoptosis Detection Kit (Abcam) according to the manufacturer's protocol. Briefly, after transfection for 48 h, cells were harvested and 500
After transfection for 48 h, cells were stained with Hoechst33342 (10 mg/ml) at room temperature in the dark for 20 min. Cells were washed with PBS and evaluated using a fluorescence microscope.
Proteins extracted from hPASMCs using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Nanjing, China) were subjected to western blotting. Proteins were quantified using a Micro BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Total cell lysates (30
Wound-healing assay was performed to evaluate the migration ability of hPASMCs. Briefly, cells (6×105 cells/well) were seeded in a 6-well plate and grown to 80-90% confluence. After aspiration of the medium, the center of cell monolayer was scraped with a yellow pipette tip to create a denuded zone of constant width. Wound closure was photographed at 0, 24 and 48 h with a Nikon inverted microscope (Nikon Corporation, Tokyo Japan).
The transwell migration assay was performed using transwell chambers with 10 mm diameter and 8
The wild-type or mutant 3′ untranslated region (UTR) of TLR4 was cloned into the pGL3 vector (Promega Corporation, Madison, WI, USA), to confirm direct target association. The wild-type contained binding sites of TLR4 3′UTR with miR-760. Cells (2×104) in each well were mixed in medium with 10% FBS and incubated for 48 h. miR-760 or negative precursor control were transfected into cells by employing Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc). After 24 h,
Cells were plated in 24-well culture plates at 1×104 cells/well. After incubation for 12 days at 37°C, cells were fixed with 4% paraformaldehyde, and stained in 10% crystal violet. The number of colonies containing ≥50 cells was counted under a microscope.
TUNEL assay was performed using an
All quantitative data were expressed as the mean ± standard deviation (n=3). GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform all statistical analysis. When only two groups were compared, Student's t-test was conducted. One-way analysis of variance followed by Tukey's post-hoc test was applied to compare differences between multiple groups. For correlation of miR-760 and TLR4 expression, the data was analyzed using Spearman's correlation. P<0.05 was considered to indicate a statistically significant difference.
Uncontrolled cell proliferation and reduced apoptosis of hPASMCs are the predominant factors of pulmonary remodeling (
The expression of miR-760 was demonstrated to be down-regulated in PAH tissues and hypoxia-induced hPASMCs, suggesting that miR-760 may function in regulating the proliferation phenotype of the pulmonary vasculature. To examine the functional role of miR-760, hPASMCs were transfected with miR-760 mimics, which resulted in a ~45% increase of miR-760 levels in hypoxia-induced hPASMCs (
Decreased apoptotic activity has been reported in hPASMCs from PAH patients (
Both PASMC proliferation and migration contribute to pathogenic pulmonary vascular remodeling in PAH (
To gain novel insights into the molecular mechanisms underlying the function of miR-760 in modulating hPASMCs, the target of miR-760 was identified. TargetScan, a miRNA prediction software, was employed (
To investigate the effect of TLR4 on cell proliferation, the expression of TLR4 was silenced with siRNA. RT-qPCR and western blot analyses were performed to assess the transfection efficiency, and the results revealed that the mRNA and protein levels of TLR4 significantly decreased in the siRNA TLR4 group compared with the siRNA NC group (
The above results suggested that TLR4 is a direct target of miR-760, and that TLR4 inhibition could decrease cell proliferation and migration, as well as induce cell apoptosis in hypoxia-induced hPASMCs. Next, RT-qPCR and western blot experiment were performed to explore the expression of TLR4 in PAH lung tissues. The data confirmed that mRNA and protein levels of TLR4 were upregulated in PAH lung tissues compared with normal lung tissues (
PAH is a chronic progressive and fatal disease (
As a well-known cause of PAH in patients, hypoxia has been widely used to generate animal or cell models of PAH (
Toll-like receptors are an important class of proteins involved in nonspecific immunity, which also link nonspecific immunity and specific immunity. TLR4, a germline-encoded pattern recognition receptor, serves a vital function in inflammatory responses (
In conclusion, the present study revealed that miR-760 may have an essential role in PAH, via regulation of TLR4. Upregulation of miR-760 drastically inhibited hPASMC proliferation and migration, as well as induced hPASMC apoptosis under hypoxic conditions, by targeting TLR4. Further studies focusing on the functional interaction between miR-760 and TLR4 might contribute to the development of novel therapeutic targets for PAH.
The present study was supported by Nanjing Medical Science and Technique Development Foundation (grant nos. 201405013 and YKK15136).
The analyzed datasets generated during the study are available from the corresponding author on reasonable request.
YZY, YFZ and WP conceived and designed the experiments. LY and JX performed the experiments. XMM analyzed the data. YZY and WP wrote the paper. All authors read and approved the final manuscript.
The experimental protocols were approved by the Ethics Committee of Nanjing Medical University, and informed consent was obtained from all the participants prior to the study.
Not applicable.
The authors declare that they have no competing interests.
Not applicable.
miR-760 is downregulated in PAH lung tissues and hypoxia-induced hPASMCs. (A) Expression levels of miR-760 in PAH and normal lung tissues. (B) Immunofluorescence staining analysis for the expression of hPASMC-specific markers (magnification, ×400). (C) Expression levels of miR-760 in hypoxia-induced and in control normoxic hPASMCs. *P<0.05 vs. the control. PAH, pulmonary arterial hypertension; hPASMC, human pulmonary artery smooth muscle cell; GPR91, succinate receptor 1.
miR-760 regulates hypoxia-induced hPASMC proliferation. (A) Expression levels of miR-760 in hypoxia-induced hPASMCs following transfection with control or miR-760 mimics. (B) Cell Counting Kit-8 assay was performed to test cell proliferation in hypoxia-induced hPASMCs transfected with miR-760 mimics. (C) Ki-67 staining in hypoxia-induced hPASMCs transfected with miR-760 mimics (magnification, ×200). (D) EdU incorporation assay in hypoxia-induced hPASMCs transfected with miR-760 mimics (magnification, ×200). Normoxic cells were used as the control group. Untransfected hypoxic cells were the blank group. #P<0.05 vs. control; *P<0.05 vs. blank. hPASMC, human pulmonary artery smooth muscle cell; NC, negative control.
miR-760 regulates hypoxia-induced hPASMC apoptosis. (A) Flow cytometry was performed to examine apoptosis in hypoxia-induced hPASMCs. (B) Hoechst 33342 staining was used to examine morphological changes related to apoptosis (magnification, ×400). (C) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was performed to evaluate hypoxia-induced hPASMC apoptosis (magnification, ×200). (D) Western blotting was performed to examine the expression levels of apoptosis-related proteins. #P<0.05 vs. control; *P<0.05 vs. blank. hPASMC, human pulmonary artery smooth muscle cell; NC, negative control; PI, propidium iodide; FITC, fluorescein isothiocyanate; Bax, BCL2 associated X; Bcl-2, BCL2 apoptosis regulator.
miR-760 modulates hypoxia-induced hPASMC migration. Cell migration was assayed in hypoxia-induced hPASMCs following transfection with miR-760 mimics, by (A) wound-healing assay (magnification, ×100), and by (B) transwell assay (magnification, ×200). #P<0.05 vs. control; *P<0.05 vs. blank. hPASMC, human pulmonary artery smooth muscle cell; NC, negative control.
TLR4 is a direct target of miR-760 in hypoxia-induced hPASMCs. (A) Binding between miR-760 and the TLR4 3′ UTR was predicted by TargetScan software analysis. (B) Luciferase reporter assays were performed to confirm the binding of miR-760 in the 3′ UTR of TLR4. (C) Western blot analysis and (D) reverse transcription-quantitative polymerase chain reaction assay were performed to detect the protein and mRNA levels of TLR4 in hPASMCs. #P<0.05 vs. control; *P<0.05 vs. blank. TLR4, toll-like receptor 4; hPASMC, human pulmonary artery smooth muscle cell; UTR, untranslated region; WT, wild-type; MUT, mutant; NC, negative control.
TLR4 silencing suppresses hypoxia-induced cell proliferation and induces apoptosis. (A) Silencing of TLR4 by siRNA was confirmed at the mRNA and the (B) protein level. (C) mRNA expression levels of TLR4 in hypoxia-induced hPASMCs following TLR4 siRNA transfection. (D) Colony formation assay in hypoxia-induced hPASMCs (magnification, ×200). (E) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was performed to examine cell apoptosis in hypoxia-induced hPASMCs (magnification, ×200). (F) Transwell assays (magnification, ×200) and (G) wound-healing assays (magnification, ×100) were performed to examine the migration ability of hypoxia-induced hPASMCs. (H) Western blot and (I) reverse transcription-quantitative polymerase chain reaction analyses were used to detect the mRNA and protein expression levels, respectively, of TLR4 in PAH and normal lung tissues. (J) Correlation analysis of miR-760 and TLR4 expression in PAH lung tissues. #P<0.05 vs. control; *P<0.05 vs. blank. TLR4, toll-like receptor 4; si, small interfering; hPASMC, human pulmonary artery smooth muscle cell; PAH, pulmonary arterial hypertension; NC, negative control.