Contributed equally
Pulmonary arterial hypertension (PAH), is a chronic and progressive disorder characterized by pulmonary vascular remodeling, including endothelial cell dysfunction and inflammation. MicroRNAs (miRNAs or miRs) play an important role in the development of PAH. In addition, fibroblast growth factor 21 (FGF21) has been found to have marked anti-dysfunction and anti-inflammatory properties. Therefore, the present study aimed to investigate the latent effects of FGF21 against PAH through the miR-27b/peroxisome proliferator-activated receptor γ (PPARγ) axis. Human pulmonary arterial endothelial cells (HPAECs) subjected to hypoxia were used as PAH models. The results revealed that PPARγ expression was downregulated and miR-27b expression was upregulated in the HPAECs exposed to hypoxia. Luciferase assay suggested that PPARγ was a target gene of miR-27b. Furthermore, miR-27b inhibited the expression of the PPARγ gene, thereby aggravating hypoxia-induced HPAEC dysfunction. Moreover, miR-27b activated the nuclear factor-κB signaling pathway and the expression of inflammatory factors [interleukin (IL)-1β, IL-6 and tumor necrosis factor-α] by targeting PPARγ. In addition, the expression of miR-27b decreased following treatment of the hypoxia-exposed HPAECs with FGF21. Furthermore, FGF21 alleviated hypoxia-induced HPAEC dysfunction and inflammation by inhibiting miR-27b expression and thereby promoting PPARγ expression. On the whole, the findings of the present study suggest that FGF21 may serve as a therapeutic target for managing PAH through the miR-27b-mediated PPARγ pathway.
Pulmonary arterial hypertension (PAH) is a chronic and progressive cardiovascular disease with a high mortality rate. It is mainly characterized by pulmonary vascular remodeling, endothelial cell (EC) dysfunction, apoptosis and inflammation. These features increase pulmonary vascular resistance and subsequent pulmonary arterial pressure, causing right heart failure and mortality (
Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear hormone receptor and transcription factor that regulates multiple genes in cardiovascular homeostasis (
MicroRNAs (miRNAs or miRs) are a conserved class of small non-coding RNAs that can regulate gene expression at the post-transcriptional level (
Fibroblast growth factor 21 (FGF21) is a member of the FGF superfamily, which is mainly expressed in the liver, pancreas, testis and brown adipose tissue. As a hormone, FGF21 regulates a variety of pharmacological effects, including blood glucose and lipid metabolism (
Pioglitazone (a PPARγ agonist) was obtained from Sigma-Aldrich; Merck KGaA. FGF21 was obtained from PeproTech, Inc. Rabbit antibodies against PPARγ (cat. no. ab178860) and NF-κB p65 (cat. no. ab32536) were purchased from Abcam). Rabbit antibodies against p-NF-κB (cat. no. 3033T) and β-actin (cat. no. 4970S) were purchased from Cell Signaling Technology, Inc. A horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (cat. no. BL003A) was obtained from Biosharp Life Sciences. Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H+L; cat. no. PC02A) was obtained from Shanghai Boyun BioTech Co., Ltd. Alexa Fluor 594 AffiniPure goat anti-rabbit IgG (H+L; cat. no. 33112ES60) was purchased from Shanghai Yeasen Biotechnology Co., Ltd. Endothelial cell medium (ECM; 1001), fetal bovine serum (FBS) and endothelial cell growth supplement (ECGS) were purchased from ScienCell Research Laboratories, Inc. Phosphate-buffered saline was purchased from HyClone; Cytiva. SuperSignal (R) West Femto Maximum Sensitivity Substrate, radio immunoprecipitation assay (RIPA) buffer, protease and phosphatase inhibitor mini tablets, and the bicinchoninic acid protein assay kit were purchased from Pierce; Thermo Fisher Scientific, Inc. The antifade-4, 6-diamidino-2-phenylindole (DAPI) was purchased from Beijing Solarbio Science & Technology Co., Ltd. The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. The ELISA kit was purchased from Shanghai Boyun BioTech Co., Ltd.
Primary HPAECs (cat. no. 3100) were obtained from ScienCell Research Laboratories, Inc. For the use of primary cells, the present study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China; approval no. 2020-232). They were cultured in ECM supplemented with 5% FBS, 1% ECGS, 100
The small interfering RNAs (siRNAs) targeting PPARγ (si-PPARγ) with the negative control (si-NC), miRNAs for miR-27b overexpression (miR-27b mimics), respective NC (mimics NC), miR-27b knockdown (miR-27b inhibitors) and respective NC (inhibitors NC) were designed and synthesized by Guangzhou RiboBio Co., Ltd. The Ribo FECT. CP transfection kit (Guangzhou RiboBio Co., Ltd.) was used to transfect the siPPARγ (50 nM), si-NC (50 nM), inhibitor (100 nM), inhibitors NC (100 nM), mimic (50 nM) and mimics NC (50 nM) into the cells following the manufacturer's protocol. Following transfection for 24 h, the cells were used in further experiments.
Following treatment, the HPAECs were lysed with ice-cold RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) for 30 min. The lysates were then centrifuged at 16,000 × g for 30 min at 4°C, and the supernatant was collected. The protein concentrations were determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein (20
Total RNA was isolated from the treated HPAECs using TRIzol reagent (Sangon Biotech). Reverse transcription was conducted using the Takara PrimeScript kit (cat. no. RR036A; Takara Bio, Inc.) at 37°C for 15 min. Subsequently, qPCR was performed using the Real-Time PCR System (cat. no. A25742; Thermo Fisher Scientific, Inc.) following the manufacturers' instructions. The PCR amplification reaction were as follows: 95°C for 10 min, 95°C for 15 sec, 62°C for 30 sec, and 72°C for 30 sec. Relative quantification was calculated using the 2−ΔΔCq method, as previously described (
TargetScan (
IF assays were conducted to determine the protein expression levels of PPARγ and NF-κB. HPAECs were seeded at a density of 1.0×105 cells per well of a 6-well plate in 2 ml of growth medium. Following stimulation, HPAECs were fixed with 4% paraformaldehyde for 30 min at 37°C and permeabilized using 0.1% Triton X-100 for 10 min. The cells were then blocked using 5% bovine serum albumin for 30 min at 37°C and immunostained using anti-PPARγ (1:200) and anti-NF-κB p65 (1:200) antibodies overnight at 4°C, followed incubation at 24°C in the dark for 1 h with 1:200 Alexa Fluor 488 conjugated donkey anti-rabbit IgG (H+L) and Alexa Fluor 594 AffiniPure goat anti-rabbit IgG (H+L). For mounting, DAPI was added to the coverslips at 24°C for 5-10 min. Images were acquired using a fluorescence microscope (Leica DMi8; Leica Microsystems, Inc.). Quantitative analysis was performed using ImageJ analysis software (v 1.51, National Institutes of Health). Each condition was repeated three times per experiment.
CCK-8 assay was performed to evaluate the viability of the HPAECs. HPAECs were seeded in 96-well plates at a density of 1×104 cells/well. Following pre-incubation in complete medium at 37°C in the presence of 21% O2 and 5% CO2 for 12-24 h, HPAECs were treated with FGF21, miR-27b mimics, miR-27b inhibitor, siPPARγ and pioglitazone prior to exposure to hypoxia. Following 24 h of exposure to hypoxia, the HPAECs were treated with 10
Cell migration assay was performed to determine the migration of HPAECs. In the present study, 24-well Transwell system (5
The levels of accumulated ET-1, TNF-α and IL-1β in the culture medium were determined using ELISA kits (ET-1, cat. no. BP-E10711; IL-1β, cat. no. BP-E10081; IL-6, cat. no. BP-E10140; TNF-α, cat. no. BP-E10110) following the manufacturer's protocol.
GraphPad Prism (v7.0; GraphPad Software, Inc.) was used for the statistical analysis of the experimental data. All data are presented as the mean ± SD of at least three independent experiments. The differences among multiple groups were analyzed using one-way ANOVA with repeated measures followed by Tukey's post-hoc test, while differences between two groups were analyzed using an unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
HPAECs were cultured under hypoxic conditions for 24 h to examine the changes in PPARγ and miR-27b expression in response to hypoxia. The results of western blot analysis demonstrated that PPARγ expression was significantly decreased in the H group compared with the N group (
Based on a bioinformatics database TargetScan, miR-27b was predicted to target PPARγ mRNA (
Subsequently, the present study explored the regulatory effects of miR-27b on PPARγ. PPARγ siRNA was used to downregulate the expression of PPARγ (
Furthermore, the results of immunofluorescence staining indicated that the fluorescence intensity of PPARγ was markedly downregulated in the H group compared with the N group. However, the fluorescence intensity of PPARγ was significantly upregulated in the H + I and H + Pio groups compared with the H group. The fluorescence intensity of PPARγ was decreased in the H + I + siPPARγ group compared with the H + I group (
Transwell assay was used to determine relative cell numbers and viability so as to assess hypoxia-induced HPAEC cell migratory ability and its regulation by miR-27b. The migratory ability of the HPAECs was decreased in the H group compared with the N group, and was markedly increased in the H + I and H + Pio groups compared with the H group, suggesting that the downregulation of miR-27b and the overexpression of PPARγ had a similar function in repairing the cell migratory ability. However, a significant reduction in the number of migrated cells was observed in the H + I + siPPARγ group compared with the H + I group (
ET-1 is a marker of endothelial dysfunction. The secretion of ET-1 was increased by hypoxia in the H group compared with the N group. ET-1 secretion was significantly decreased in the H + I and H + Pio groups compared with the H group; however, the secretion of ET-1 was upregulated in the H + I + siPPARγ group (
The relative levels of p-NF-κB and NF-κB expression in the different groups of HPAECs were determined by western blot analysis to elucidate the molecular mechanisms underlying the effects of miR-27b on PAH-related inflammation. The results revealed that the ratio of p-NF-κB/NF-κB was markedly increased in the H group compared with the N group. The ratio of p-NF-κB/NF-κB was decreased in the H + I and H + Pio groups compared with the H group; however, an adverse trend was observed in the H + I + siPPARγ group (
Subsequently, the levels of inflammatory factors, such as IL-1β, IL-6 and TNF-α, as secreted cytokines, were examined by ELISA, as this may better reflect their expression in the medium. The results indicated that markedly higher levels of IL-1β, IL-6 and TNF-α were found in the culture medium of HPAECs exposed to hypoxia. The levels of IL-1β, IL-6 and TNF-α were significantly decreased in the H + I and H + Pio groups, whereas this decreasing tendency was reversed in the H + I + siPPARγ (
The present study then examined the effect of FGF21 on the miR-27/PPARγ axis. Firstly, it was found that the expression of FGF21 was decreased in the H group compared with the N group (
Furthermore, CCK-8 and Transwell assays, as well as ELISA were used to examine the effect of FGF21 on the dysfunction of hypoxia-exposed HPAECs via the miR-27b/PPARγ axis. FGF21 markedly promoted cell viability and migration under hypoxic conditions; however, this change was abolished in the H + F + m group (
HPAECs were treated with FGF21 prior to exposure to hypoxia to further examine the functional antagonistic effect of FGF21 on hypoxia-induced inflammation. The results of western blot analysis indicated that the ratio of p-NF-κB/NF-κB was markedly decreased in the H + F group compared with the H group. However, this change was reversed in the H + F + m group (
PAH is a vascular disorder with a high morbidity and mortality due to the limited treatment methods available. Recent studies have indicated that PAH os closely related to endothelial dysfunction and inflammation (
Previously, PPARγ was reported to reduce ET-1 and endothelial dysfunction in sickle cell disease-associated PH (SCD-PH) (
Recently, miR-27b has been suggested to be a key biomarker in various diseases (
FGF21, as an important endocrine regulator, has recently been reported to prevent angiotensin II-induced hypertension and vascular dysfunction in mice (
In conclusion, the findings of the present study suggested that miR-27b exacerbated hypoxia-exposed HPAEC dysfunction and inflammation by inversely regulating PPARγ. However, FGF21 abolished the effect of miR-27b on hypoxia-exposed HPAECs, mainly by inhibiting miR-27b expression, thereby promoting the expression of PPARγ. Due to limited conditions, the role of the FGF21/miR-27b/PPARγ axis
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
DY and QH designed and performed the experiments. QH analyzed the data and wrote the manuscript. XH, LW and YL designed the experiments and assisted in the drafting of the manuscript. JS, LC and JW performed the experiments and collected the data. GC and JL collected the data and performed the analysis. DY and QH confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
The present study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) (approval no. 2020-232).
Not applicable.
The authors declare that they have no competing interests.
Not applicable.
miR-27b suppresses PPARγ expression by targeting the PPARγ gene. (A) Protein expression levels of PPARγ were determined by western blot analysis. (B) Expression of miR-27b was determined by RT-qPCR. (C) Predicted binding sites of miR-27b matching the 3′-UTR of PPARγ. The luciferase activity was decreased following treatment with a combination of miR-27b mimic and PPARγ-3′-UTR-WT, suggesting that miR-27b regulated PPARγ. (D) Western blot analysis to determine PPARγ siRNA efficiency. (E and F) HPAECs were transfected with inhibitors NC or miR-27b inhibitors, mimics NC or miR-27b mimics, and at 24 h following transfection, the expression of miR-27b was determined by RT-qPCR. (G and H) Western blot analysis of PPARγ protein levels in the different groups. β-actin was used as a loading control. The measurement data are presented as the mean ± standard deviation and analyzed by one-way analysis of variance. The experiment was performed in triplicate. *P<0.05; **P<0.01; ***P<0.001, vs. the respective control. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; WT, wild-type; MUT, mutant-type; UTR, untranslated region; NC, negative control; N, normoxia + NC group; H, hypoxia + NC group; PPARγ, peroxisome proliferator-activated receptor γ; HPAECs, human pulmonary arterial endothelial cells.
miR-27b aggravates dysfunction of hypoxia-exposed HPAECs by targeting PPARγ. (A and B) Expression of PPARγ (green) was detected by immunofluorescence staining, and the fluorescence intensity of PPARγ was calculated; DAPI was used to stain cell nuclei (blue). Scale bars, 50
miR-27b pomotes hypoxia-induced HPAEC inflammation by targeting PPARγ. (A) Protein levels of p-NF-κB and NF-κB were detected by western blot analysis. β-actin was used as a loading control. (B and C) Immunofluorescence staining was used to detect NF-κB (red) entering the nucleus. DAPI was used to stain cell nuclei (blue). Scale bars, 100
FGF21 attenuates hypoxia-induced dysfunction of HPAECs through the miR-27b/PPARγ axis. (A) Protein levels of of FGF21 was determined by western blot analysis. β-actin was used as a loading control. (B) RT-qPCR was used to determine the expression of miR-27b. (C and D) The expression of PPARγ (green) was detected by immunofluorescence staining, and the fluorescence intensity of PPARγ was calculated. DAPI was used to stain cell nuclei (blue). Scale bars, 50
FGF21 suppresses the NF-κB signaling pathway and the inflammatory response in hypoxia-exposed HPAECs via the miR-27b/PPARγ pathway. (A) Protein levels of p-NF-κB and NF-κB were detected by western blot analysis. β-actin was used as a loading control. (B and C) Immunofluorescence staining was used to detect NF-κB (red) entering the nucleus. DAPI was used to stain cell nuclei (blue). Scale bars, 100