Data collection and analysis of differentially expressed genes
The method we used is similar to that reported in an earlier study we published. 19 Briefly, the gene expression profiling datasets GSE2498820, GSE11343921 and GSE11726122 are based on GPL6244 ([HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version]) were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The GSE24988 dataset contained 62 PH and 22 normal lung tissues. The GSE113439 dataset contained 15 PH and 11 normal lung tissues. The GSE117261 dataset contained 58 PH and 25 normal lung tissues. The 3 datasets were merged and normalized using the “sva” R package. We identified differentially expressed genes (DEGs) using the “limma” package in R. Values with P < 0.05 and |log2Fold change (logFC)| >0.5 were considered statistically significant.
HPH rat model
H19-deficient rats (H19−/−) on a Sprague Dawley background were purchased from Cyagen Biosciences Inc. and bred in our animal feeding room under controlled conditions (12 h light/dark cycle, 23 ± 2°C). After that, the tails of the offspring rats were isolated and genotyped by PCR (Figure S1A-B). Eight-week-old male homozygous rats and their wild-type (WT) littermates were used in the experiment.
To induce HPH, rats were placed in an atmospheric hypoxia incubator (Changjin, Changsha) at 10% O2, and the control rats were maintained in normoxic conditions for 4 weeks. All experimental procedures involving rats were carried out during the study following the principles approved by the University of Xinxiang Animal Care and Use Committee.
Echocardiographic assessment
After 4 weeks of hypoxia treatment, the rats were anaesthetized with isoflurane (2%) and imaged with a VEVO 2100 imaging system (Visual Sonics, Ontario, Canada) equipped with a 30 MHz probe. Stable images were obtained in M and Doppler modes, and the acceleration time (PAAT) and ejection time (PAET) of the pulmonary artery and tricuspid annular plane systolic excursion (TAPSE) were measured.
Right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) measurement
The rats were anaesthetized, and their right external jugular veins were stripped and slit. Then, the PE catheter filled with heparin saline and connected with a pressure transducer (TaiMeng, Chengdu, China) was slowly inserted into the blood vessel from the incision. The right ventricular end systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) was recorded in real time after the pressure waveform stabilized.
Sampling
After RVSP measurement, all rats were sacrificed under anaesthesia, and the heart samples were removed. The ratio of right ventricle to left ventricle plus ventricular septum (RV/LV + S) and right ventricular weight to tibial length (RV/TL) were used as indices of right ventricular hypertrophy. Meanwhile, the lung tissue and secondary branches of the pulmonary artery of all rats were collected. A portion of the lung tissue samples was stored at -80 ℃, and the other lung tissue was soaked in 4% paraformaldehyde solution. The pulmonary arteries were kept in precooled electron microscope fixative (2.5% glutaraldehyde) at 4 ℃ overnight.
Transmission electronic microscope (TEM)
The pulmonary arteries immersed in 2.5% glutaraldehyde were trimmed and fixed in 1% osmic acid fixative for 3 h. After dehydration with gradient alcohol and soaking in Embed 812 (14120, SPI, USA) overnight, all pulmonary arteries were baked and solidified in an oven at 60 ℃ for 48 h and subsequently cut into 70-nm slices. Afterwards, the slices were stained with 3% uranium acetate lead citrate and observed and photographed by transmission electron microscopy (Phillips, Netherlands).
Morphological staining
Lung tissues were fixed with 4% paraformaldehyde for 16 h, dehydrated with gradient alcohol, and subsequently embedded in paraffin. Then, the tissues were sliced into 4-µm sections, stained with haematoxylin and eosin stain (HE), Masson or van Gieson (VG) as per the standardized protocols, and observed by light microscopy (Olympus, Japan).
In situ hybridization (ISH)
An in situ hybridization (ISH) assay was conducted using an RNA ISH Kit (GDP1061, Servicebio, Wuhan, China) according to the manufacturer’s protocol. Briefly, paraffinized lung tissue sections were exposed to mRNA fragments using citric acid and Protease K, endogenous peroxidase activity was blocked with 3% H2O2, and the sections were reacted with prehybridization solution at 37 ℃ for 1 h. Then, the slices were incubated in H19 probe hybridization solution at 42°C overnight. After rinsing with SSC three times, the slices were incubated in hybridization solution with a secondary probe and blocked with 3% FBS for 30 min. Next, the sections were reacted with anti-DIG-HRP for 50 min, washed with PBS, and developed with DAB for 10 min. Finally, the sections were counterstained with haematoxylin for 3 min and observed under an optical microscope (Olympus, Japan). The sequence of the H19 probe was 5’-GGGCTAGAGGCTTGGCTCCAGGATGATGT (ttt CATCATCAT ACATCATCAT) 30 − 3’, and the sequence of the secondary probe was 5’-DIG-tt-ATGATGATGT ATGATGATGT-3’.
Hypoxia-induced HPH in PAECs
Primary rat pulmonary artery endothelial cells (RPAECs) were isolated from pulmonary arteries by the collagenase digestion method and then enriched by magnetic sorting. Briefly, male Sprague‒Dawley rats (200–350 g) were sacrificed, and their pulmonary arteries were excised. The pulmonary arteries were cut thoroughly and digested with collagenase I for 1 hour. After 200 mesh cell sieve filtration, CD31-FITC was added to the cell suspension, and RPAECs were enriched by magnetic sorting. The cells were maintained in EBM-2 (Lonza) supplemented with 10% fetal bovine serum (FBS) (HyClone) at 37°C in the presence of 5% CO2. Primary cells were allowed to grow and were passaged at confluency by trypsin digestion into culture flasks. RPAECs were characterized by indirect immunofluorescence using an antibody specific to rat CD31 and α-SMA (Figure S2). We carried out follow-up experiments with cells within 5 generations.
human pulmonary artery endothelial cells (HPAECs) purchased from XinYu Biotechnology Inc. (XY-h443, Shanghai, China) were grown in special culture medium for endothelial cells (CP0028, XinYu) containing 10% FBS (WGG8001-100, Servicebio, Wuhan, China) at 37°C.
To establish an HPH model in PAECs, RPAECs or HPAECs that reached 50% confluence were starved with medium with a serum concentration of 0.02% for 12 h and then cultured in an anoxic incubator (3% O2) for 48 h.
Cell transfection
Small interfering RNA (siRNA) oligonucleotides for H19 (H19-si), TGFβR1 (TGFβR1-si), siRNA negative control (NC-si), miR-let-7g-5p mimic and mimic negative control (mimic-NC), miR-let-7g-5p inhibitor and inhibitor negative control (inhibitor-NC) were generated by RiboBio Biotechnology Inc. (Guangzhou, China). Cell transfection was performed in PAECs via a riboFECT CP Transfection Kit (C10511-05, RiboBio) in accordance with the manufacturer's protocol. After 12 h, the transfected cells were collected and subsequently treated with hypoxia.
Immunofluorescence
After transfection and chronic hypoxia treatment, the HPAECs cultured on sterilized coverslips were fixed with 4% paraformaldehyde for 20 min. Then, the cells and lung sections were reacted with 0.5% Triton X-100 and blocked with 3% FBS (WGG8001-100, Servicebio) at room temperature for 1 h. Afterwards, the cells and sections were incubated with primary antibodies against CD31 (1:1,000 dilution; cat. no. ab9498; Abcam) or α-SMA (1:50 dilution; cat. no. ab150301; Abcam) overnight at 4°C and subsequently counterstained with the FITC/Cy3-conjugated secondary antibody (1:400 dilution, A11008, affinity). After rinsing with PBS three times, the coverslips and tissue sections were stained with DAPI for 5 min at room temperature. The results were imaged using a fluorescence microscope (Olympus, Japan).
Dual luciferase reporter assay
The sequences of H19 containing wild-type (WT) or mutant (MUT) let-7 g-5p binding sites were generated and cloned into the pmiR-RB-Report™ luciferase reporter vector (Ribobio), generating corresponding constructs H19-WT and H19-MUT. Similarly, the 3′UTR of TGFβR1 containing let-7 g-5p binding sites or its corresponding mutant was used to generate TGFβR1-WT and TGFβR1-MUT on the basis of the pmiR-RB-Report™ luciferase reporter vector. For the dual luciferase reporter assay, HEK293T cells were cotransfected with let-7g-5p mimic or mimic-NC and constructed vectors by lipofectamine™ 3000. After 48 h, luciferase activity analysis was performed using an ONE-Glo™ EX Luciferase Assay System (E8110, Promega, USA), with Renilla luciferase activity as a control.
qRT‒PCR
Total RNA from pulmonary arteries or PACEs was extracted by using TRIzol (Invitrogen, ON, Canada). Then, the cDNA for PCR was produced using 2 µg total RNA and KEIris RT mix with dsDNase (All-in-One) (Codonx, Beijing, China). Next, RNA expression levels were evaluated by using TB Green® Premix Ex Taq™ II Kit (Takara, Japan) on an Azure CieloTM real-time PCR system (Azure Biosystems, USA). The specific primers were designed using primer 3 and are listed as follows:
h-H19-Forwards: 5’-CGTGACAAGCAGGACATGACA-3’
h-H19-Reverse: 5’-CCATAGTGTGCCGACTCCG-3’
r-H19-Forwards: 5’-CTAAGTCGATTGCACTGGTTTGG-3’
r-H19-Reverse: 5’-ACACCCAGTTGCCCTCAGAC-3’
Western blot
The total protein content was extracted by precooled lysis buffer and quantified by bicinchoninic acid (BCA) protein assay kit (WB6501, New Cell & Molecular Biotech LTD, Suzhou, China). Subsequently, equal samples containing 30 µg proteins were electrophoretically separated via 10% SDS‒PAGE and then blotted onto polyvinylide fluoride (PVDF) membranes. The proteins were blocked with blocking buffer (P30500, New Cell & Molecular Biotech) for 15 min and then incubated overnight at 4°C with primary antibodies against CD31 (AF6191, 1:1000), α-SMA (AF1032, 1:1000), Vimentin (AF7013, 1:1000), TGFβR1 (AF5347, 1:1000) and β-actin (AF7018, 1:10000). The membranes were incubated in the HRP-linked anti-rabbit IgG secondary antibody (S0001, 1:6000) for 1 h. All the above mentioned antibodies were purchased from Affinity Biosciences LTD (CA, USA). Finally, the membrane was immunostained with a Bio-Rad image analysis system (Bio-Rad Inc., CA, USA) using an ECL kit (P2300, New Cell & Molecular Biotech).
Statistical analysis
The data are presented as the means ± SEMs, and significant differences among groups were analysed using the unpaired t test (two groups) or one-way ANOVA test (more than two groups) in Statistical Product and Service Solutions (SPSS) 19.0 (Systat Software, San Jose, CA, USA). P < 0.05 was deemed statistically significant.