Forty patients with idiopathic PAH were consecutively enrolled from January 2017 to March 2018 at the Guangdong Provincial People's Hospital. Their clinical diagnosis of idiopathic PAH was according to the 2015 ESC/ERS guidelines for the diagnosis and treatment of pulmonary hypertension . The exclusion criteria were:
Initiation of PAH-specific therapies before enrollment
Severe liver and kidney dysfunction
Other acute or chronic fatal diseases that could definitely worsen the prognosis
Inability to sign informed consent or cooperate with the researchers
Twenty normal volunteers matched for age, gender and race were enrolled simultaneously. Written informed consent was signed by all participants. The study was approved by the Ethics Committee of the Guangdong Provincial People’s Hospital (No. GDREC2016305H).
Right-side heart catheterization was performed following admission. The values of the mean pulmonary arterial pressure, pulmonary vascular resistance, and pulmonary vascular resistance index of the PAH patients were recorded. Plasma was collected and stored at – 80 °C for subsequent assays.
RNA extraction and quantitative RT-PCR
A HiPure Liquid RNA/miRNA Kit (R4163-02; MAGEN, China) was used to extract total RNA from the plasma. The RNA (1 µg) was reverse transcribed using MMLV Reverse Transcriptase (RT; Promega, USA). The RT primer for miR-361-3p was 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAATCAGA-3′ and for the internal reference gene U6 was 5′-AACGCTTCACGAATTTGCGT-3′. The qPCR mix was prepared using SYBR Green qPCR SuperMix (Invitrogen, USA) and qPCR was performed on an ABI PRISM 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, USA). The forward primer for miR-361-3p was 5′-ACACTCCAGCTGGGTCCCCCAGGTGTGATTCTG-3′ and the reverse primer was 5′-CTCAACTGGTGTCGTGGA-3′. The forward primer for U6 was 5′-CTCGCTTCGGCAGCACA-3′ and the reverse primer was 5′-AACGCTTCACGAATTTGCGT-3′. The relative expression of miR-361-3p was calculated using the 2−ΔΔCt method .
Human PASMCs (hPASMCs) were purchased from ScienCell Research Laboratories (USA) and cultured in smooth muscle cell medium (cat. no. 1101, ScienCell Research Laboratories) at 37 °C in a humidified incubator infused with air (21% O2 and 5% CO2).
Preparation of the miR-361-3p mimic and construction of the serotonin transporter (SERT) overexpression plasmid
Negative control miRNA (miR-NC), miR-361-3p mimic (miR-361-3p), miR-NC inhibitor and miR-361-3p inhibitor were purchased from GenePharma (China). To construct the SERT overexpression plasmid (ov-SERT), the full coding sequence of SERT was cloned into plasmid pcDNA3.1 + at the KpnI and XhoI sites, using the following primers: 5′-cggggtaccgccaccATGGAGACGACGCCCTTGAATTCTCAG-3′ and 5′-ccgctcgagTTACACAGCATTCAAGCGGATGTCCCCACA-3′. Empty plasmid pcDNA3.1 + was used as a negative control (pCDNA).
Serotonin treatment, transient transfection and cell groups
To investigate the effect of serotonin on the proliferation of hPASMCs, cells were treated with 0, 50, 100, 250, 500 and 1000 μmol/l serotonin for 0, 24, 48 and 72 h. To investigate the effect of miR-361-3p on serotonin treated-hPASMCs, cells were divided into four groups: blank (no treatment), serotonin (treated with 250 μmol/l serotonin for 48 h), serotonin + miR-NC (transfected with miR-NC for 24 h, then treated with 250 μmol/l serotonin for 48 h), serotonin + miR-361-3p group (transfected with miR-361-3p mimic for 24 h, then treated with 250 μmol/l serotonin for 48 h).
To investigate whether SERT overexpression weakens the effect of miR-361-3p overexpression, the serotonin-treated-hPASMCs were divided into three groups: miR-NC + pcDNA (transfected with miR-NC and empty pcDNA3.1 + plasmid), miR-361-3p + pcDNA (transfected with miR-361-3p mimic and empty pcDNA3.1 + plasmid), and miR-361-3p + ov-SERT group (transfected with miR-361-3p mimic and ov-SERT). The transient transfections were performed using Lipofectamine 2000 (Invitrogen).
The CellTiter 9 AQueous One Solution Cell Proliferation Assay (MTS, Promega) was used to assess the effect of the various treatments on cell proliferation. Briefly, CellTiter 96 AQueous One Solution reagent (20 μl) was added to the culture medium at the end of the experimental period and incubated in a humidified incubator (21% O2 and 5% CO2) for 2 h. The absorbance was measured at an optical density (OD) of 490 nm using a Multiskan MK3 microplate reader (Thermo Fisher Scientific). The rate of cell proliferation was calculated as: proliferation rate = (experimental OD value – blank OD value)/(control OD value – blank OD value) × 100%.
5-ethynyl-2′-deoxyuridine (EdU) staining
Following the treatments described in the methods section, the cells were incubated in culture medium containing 50 μM EdU (Solarbio, China) for 2 h. Next, the cells were fixed by successive incubation with 4% paraformaldehyde (Merck KGaA, Germany) in phosphate-buffered saline (PBS) for 20 min, 2 mg/ml glycine (Merck KGaA) for 10 min, and 0.5% TritonX-100 (Merck KGaA) in PBS for 10 min. After washing with PBS once, 1× Apollo staining solution (Solarbio) was added to each well to detect the EdU signal and incubated for 30 min. After decolorization with 0.5% TritonX-100 in PBS, the cells were washed twice with methyl alcohol and once with PBS, and then stained with 1× Hoechst 33342 solution (Solarbio) for 30 min to stain the nuclei. After washing with PBS three times, five randomly selected fields were photographed with a fluorescence microscope (EVOS FL Auto Cell Imaging System; Thermo Fisher Scientific). The numbers of EdU-positive and Hoechst 33342-stained cells were counted using Image Pro-Plus 6.0 (Media Cybernetics, USA). The percentage of EdU-positive cells was calculated as: (number of EdU-positive cells/number of Hoechst 33342-stained cells) × 100%.
Cell cycle assay
Harvested cells were suspended in 300 μl PBS, then 700 μl absolute ethanol was slowly added to fix the cells. After fixing at – 20 °C overnight, the cell suspension was centrifuged at 2000 rpm for 10 min, and the supernatant was discarded. After washing with PBS, the cell pellet was stained with 300 μl propidium iodide solution at 37 °C in the dark for 15 min.
Total cellular protein extraction, protein concentration quantification and western blotting were performed as described in our previous report . Briefly, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane after separation using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After blocking with 5% non-fat milk (5 g/100 ml PBS containing 0.1% Tween-20) at 25 °C for 2 h, the PVDF membranes were separately incubated with primary antibodies at 4 °C overnight. The next day, the PVDF membranes were washed to remove unbound primary antibodies and then incubated with the secondary antibody. Finally, after washing to remove unbound secondary antibodies, the chemiluminescent signal was detected via exposure to X-rays. The primary antibodies used in western blotting were: anti-cyclin D1 (1:1000, ab226977), anti-cyclin E1 (1:2,000, ab71535), anti-SERT (1:800, ab102048), anti-total extracellular-regulated kinase (ERK)1/2 (t-ERK1/2, 1:1000, ab17942), anti-phosphorylated ERK1/2 (p-ERK1/2, 1:800, ab2143620), anti-cleaved caspase-9 (1:1000, ab2324), anti-Bcl-2 (1:2000, ab196495), and anti-GAPDH (1:3000, ab9485). All the primary antibodies were purchased from Abcam (USA). GAPDH was used as a loading control.
Luciferase reporter assay
miR-361-3p-binding sites in the 3′-untranslated region (UTR) of SERT messenger RNA were predicted using TargetScan (human 7.2 version, https://www.targetscan.org/). To construct the luciferase reporter, wild-type SERT 3′-UTR was cloned into the psi-CHECK-2 vector (Promega), using the following primers: 5′-ccgctcgagCACACTCACCGAGAGGAAAAAGGCTTCTCC-3′ (forward) and 5′-ataagaatgcggccgcTTCACAGCATAAATCATTTATTAATATC-3′ (reverse). PCR-based site-directed mutagenesis was performed to mutate the miR-361-3p-binding sites in wild-type luciferase reporter using the following primers: 5′-GAATTTTGTCGTTGAAAAACGAGAATAGATGGCATCAGTCCTTCAATTCTGTAACT-3′ (forward) and 5′-CATCTATTCTCGTTTTTCAACGACAAAATTCTTCTTAGTTCAGTAGACATTCAAAC-3′ (reverse). The wild-type and mutant luciferase reporters were called wild-type 3′-UTR and mutant 3′-UTR, respectively.
Human embryonic kidney 293 T cells were plated in 24-well plates and co-transfected with 0.5 µg wild-type 3′-UTR plasmid along with 50 nM miR-361-3p mimic, 0.5 μg wild-type 3′-UTR plasmid with 50 nM miR-NC, 0.5 μg mutant 3′-UTR plasmid with 50 nM miR-361-3p mimic, or mutant 3′-UTR plasmid with 50 nM miR-NC using Lipofectamine 2000. The activity of firefly luciferase or Renilla luciferase was measured 48 h post-transfection using a Dual-Luciferase Assay kit (Promega). The relative luciferase activity was expressed as a ratio of Renilla luciferase to firefly luciferase.
Anti-AGO2 RNA immunoprecipitation (RIP) assay
hPASMCs were transfected with miR-361-3p mimic or miR-NC. After transfection for 48 h, cells were harvested and resuspended in 100 µl cell lysis buffer for immunoprecipitation (Beyotime Biotechnology, China) containing protease and RNase inhibitors. The RIP assay was performed following the instructions for the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA). The antibodies anti-AGO2 and anti-IgG were purchased from Abcam. Finally, the SERT mRNA level in the RIP products was analyzed using quantitative RT-PCR. The cell lysate before immunoprecipitation was termed the ‘input’ group. The products of the RIP assay targeting AGO2 or IgG were named the AGO2 group or IgG group, respectively.
Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad software, USA). The expression levels of miR-361-3p in the plasma from PAH patients and normal controls were described using the median and the 25th and 75th percentiles. All the data pertaining to miR-361-3p expression profiles in the plasma were analyzed using the Mann–Whitney U test. Results from three independent experiments are expressed as means ± standard deviation. Statistical differences for the cell experiments were evaluated using one-way analysis of variance. p < 0.05 was considered statistically significant.