Animals and lung tissue preparation

Healthy male C57BL/6J mice with a mean weight of 30 g were obtained from the Experimental Animal Center of Harbin Medical University (Harbin, China). To confirm the role of the functional fragment TFO2 of FENDRR (464–516) in HPH, The TFO2 sequence of FENDRR cloning construction and serotype 5 adenovirus-associated virus (AAV 5) packaging experiment were constructed by HANBIO (Shanghai, China). An aliquot of the vector at 1011 genome equivalents was prepared in 20–30 μL of HBSS and isoflurane anesthesia followed by nasal drops. Mice were randomly divided into five groups as follows: normoxic environment plus control vector group (NOR + NC, n = 20), hypoxic environment plus control vector group (HYP + NC, n = 10), hypoxic environment plus FENDRR TFO2 adenovirus group (HYP + FENDRR TFO2, n = 10), normoxic environment plus FENDRR TFO2 adenovirus group (NOR + FENDRR TFO2, n = 10). Seven days later, mice were assigned to normoxia (Fi,O2 0.21) and hypoxia (Fi,O2 0.10) for seven days as previously described (Zhu et al. 2003). The mice were administered nontargeted control vector or FENDRR TFO2 adenovirus intranasally again and were assigned to normoxia (Fi,O2 0.21) and hypoxia (Fi,O2 0.10) for 14 days. All mice were anaesthetized through an intraperitoneal injection of avertin (200 mg/kg i.p., Sigma-Aldrich, St Louis, USA). For the right ventricular hypertrophy index (ratio of right ventricular free wall weight over the sum of septum plus left ventricular free wall weight: RV/(LV + Sep) calculation, hearts were excised and atria were removed. The RV free wall was dissected, and each cham-ber was weighed.

Echocardiography and right ventricular systolic pressure (RVSP) measurements

The right ventricular systolic pressure (RVSP) and echocardiography were measured as previously described (Liu et al. 2020a). The right ventricular systolic pressure (RVSP) was measured with PowerLab monitoring equipment (AD Instruments, Colorado Springs, CO). A 1.2 French Pressure Catheter (Scisense Inc, USA) was inserted into the superior vena cava and finally into the right ventricular vein, and the RVSP was continuously recorded for 20–40 min. Mice were subjected to echocardiography using a Vevo2100 imaging system (VisualSonics Inc., Toronto, Ontario, Canada), pulmonary artery velocity time integral (PAVTI), pulmonary artery acceleration time (PAAT) and left ventricular ejection fraction (LVEF) were obtained from stable images.

Morphometric analysis

Hematoxylin and eosin staining (HE staining) was performed according to the manufacturer’s instructions. In brief, lung tissues of mice were immersed in 4% paraformaldehyde for 48 h. Next, the fixed lung tissues were dehydrated, cleared and embedded in paraffin wax. The lung tissue volume of each block was sampled with equal probability. The paraffin blocks were cut into 5-μm-thick sections and stained with hematoxylin and eosin (HE). In situ hybridization was performed with kits following the manufacturer’s instructions (Boster, Wuhan, China). Digoxigenin-labeled DNA probes complementary to TFO2 of FENDRR were generated using random primer labeling. For each slice stained, 6 high power fields were randomly selected for analysis. The total wall thickness and positive staining area in the vascular walls were quantified by using a color-recognition algorithm in Image-Pro Plus 6.0 software.

Cell culture

HPAECs used in the experiment were purchased from ScienCell Research Laboratories (CA, USA). HPAECs were maintained in endothelial cell medium (ScienCell, 1001, CA, USA) containing 15% fetal bovine serum and 1% penicillin streptomycin at 37 °C, 5% CO2, and 100% relative humidity. Cells under hypoxic conditions were incubated in a Tri-Gas Incubator (Heal Force) with a water-saturated atmosphere comprising 3% O2, 5% CO2 and 91% N2 for 24 h.

Fluorescent in situ hybridization (FISH)

Fluorescence-conjugated FENDRR probes were synthesized by RuiBo (Guangzhou, China). FISH experiments were performed using a Fluorescent In Situ Kit (RuiBo Biology, Guanzhou, China) following the manufacturer’s instructions. Briefly, HPAECs were cultured on coverslips and then grown to approximately 60%. After being treated with agents according to the different experimental groups, cells were washed with 1 × PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.3% Triton X-100. Then, the cells were blocked with prehybridization solution at 37 °C for 1 h and incubated with hybridization solution containing FENDRR, 18S and U6 probes overnight at 37 °C in the dark. Finally, 4′,6-diamidino-2-phenylindole (DAPI) was added to stain the nuclei at 37 °C for 10 min. Images were captured with a living cell workstation (AF6000; Leica, Germany).

Isolation of cytoplasmic and nuclear RNAs

Cytoplasmic and nuclear RNAs were isolated and purified using a Norgen’s Cytoplasmic & Nuclear RNA Purification Kit (Thorold, ON, Canada) following the manufacturer’s instructions. In brief, 1 × 106 HPAECs were lysed with ice-cold lysis buffer, and cytoplasmic RNA and nuclear RNA were bound to the column. Finally, the mixture was separated for RNA elution analysis.

Western blot analysis

Protein samples were extracted from HPAECs by using ice-cold lysis buffer and then centrifuged at 13,500 rpm for 15 min at 4 ℃. After centrifugation, the protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). Protein samples (30 µg) were fractionated on 12% SDS-PAGE gels, transferred onto nitrocellulose membranes, and subsequently blocked with 5% nonfat milk at room temperature for 1 h. The membranes were incubated with specific antibodies against NLRP3 (2 µg/mL, bs-10021R, Bioss, Beijing, China), Caspase-1 (1 µg/mL, 22915-1-AP, Proteintech, IL, USA), pro-Caspase-1 (1 µg/mL, ab179515, Abcam, MA, USA), IL-1β (1 µg/mL, 16806-1-AP, Proteintech, IL, USA), DRP1 (1 µg/mL, ab184247, Abcam, MA, USA), YTHDC1 (2 µg/mL, 14392-1-AP, Proteintech, IL, USA), FTO (2 µg/mL, bs-7056R, Bioss, Beijing, China). Bands were sequentially incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h and enhanced chemiluminescent reagent imaging.

Quantitative RT–PCR

Total RNA was extracted from HPAECs using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The concentration and purity of all samples were measured via a NanoDrop 2000 (Thermo Scientific, Wilmington, USA), and cDNA was synthesized with the use of the Superscript first-strand complementary DNA synthesis kit (Invitrogen). Finally, the products were quantified using SYBR Green real-time PCR (Toyobo, Japan) in a Roche LightCycler 480II instrument. The nucleotide sequences of the primers are shown in Table 1.

Table 1 All primers and probes sequencesPlasmid vector constructs and transfection

For the overexpression assay, FENDRR and DRP1 plasmids were constructed using the vector GV219, and empty vector alone was used as a negative control (GeneChem, Shanghai, China). HPAECs were transfected with 3 μg of plasmids using Lipofectamine 2000 reagent following the manufacturer’s instructions. Then, 4–6 h after transfection, the cells were switched to 5% serum-containing medium and cultured under normoxic or hypoxic growth conditions for another 24 h.

siRNA, ASO design and transfection

The expression of genes was silenced by transfecting HPAECs with small interfering RNAs (siRNAs) or antisense oligonucleotides (ASOs), which were designed and synthesized by GenePharma (Shanghai, China) and RiboBio (Guangzhou, China). HPAECs were transfected with 2 μg of siRNAs or ASO using X-tremeGene siRNA transfection reagents following the manufacturer’s instructions. Six hours after transfection, the cells were switched to 5% serum-containing medium and cultured under normoxic or hypoxic growth conditions for another 24 h. The detailed siRNA sequences are shown in Table 1.

Hoechst 33342/PI fluorescent staining

HPAECs were cultured on coverslips until the cell confluence reached 80%. The cells were treated with different agents according to the different experimental groups. Afterward, the cells were stained with 6 μL of Hoechst 33342 solution and 6 μL of PI (propidium iodide) at 4 °C in the dark for 20 min. Images were captured with a living cell workstation (AF6000; Leica, Germany).

LDH release assay

HPAECs were plated into 96-well plates at a density of 5000 cells/well, and then cells were treated with different agents according to the different experimental groups. After 24 h of hypoxia, LDH was measured according to the LDH Release Assay Kit instructions (Beyotime Biotechnology, Shanghai, China). Finally, absorbance at 490 nm was recorded.

Cell and tissue immunofluorescence

HPAECs were cultured on coverslips in 12-well plates and then treated with different agents according to the different experimental groups. Prepared cells were washed three times with 1 × PBS and were fixed with 4% paraformaldehyde at 4 °C for 15 min. Then, the cell membrane was permeabilized with 0.3% Triton X-100 for 30 min and blocked with 5% bovine serum for 30 min at room temperature. After that, the cells were incubated with DRP1 (10 µg/mL, ab184247, Abcam, MA, USA), Caspase-1 (6 µg/mL, 22915-1-AP, Proteintech, IL, USA), NLRP3 (10 µg/mL, bs-10021R, Bioss, Beijing, China) and CD31 (5 µg/mL, ab9498 Abcam, MA, USA) antibodies in PBS at 4 °C overnight. After washing three times with 1 × PBS, the cells were subsequently incubated with Cy3-conjugated goat anti-rabbit (A0516, Beyotime, Shanghai, China) and FITC-conjugated goat anti-mouse (A0568, Beyotime, Shanghai, China) antibodies for 2 h at 37 °C in the dark. Cells were then washed with 1 × PBS, DAPI (C1002, Beyotime, Shanghai, China) was added to stain the nuclei at 37 °C for 10 min. Finally, the coverslips were mounted with anti-fade mounting medium (P0126, Beyotime, Shanghai, China) and captured with a living cell workstation. The frozen sections of mouse lung tissues were performed in the same manner.

RNA immunoprecipitation (RIP)

The RNA immunoprecipitation assay was performed by using an RNA Immunoprecipitation (RIP) Kit (Bes5101, BersinBio, Guangzhou, China) following the manufacturer’s instructions. Briefly, 1 × 107 HPAECs were lysed with RIP lysis buffer. After removing DNA, 20 µL of protein A/G bead-conjugated anti-YTHDC1 antibodies (3 µg, 14392-1-AP, Proteintech, IL, USA) and IgG were added to the samples and incubated overnight at 4 °C. After extracting RNA, the expression of FENDRR was detected by qRT-PCR. For m6A-RNA immunoprecipitation (Me-RIP), an anti-m6A antibody (4 µg A-1801, Epigentek Group Inc., Farmingdale, NY) was used. FENDRR extracted from cell lysates was used to measure the m6A-methylated level of FENDRR.

Chromatin isolation by RNA purification (CHIRP)

The interaction between FENDRR and the promoter of DRP1 was determined using chromatin isolation by RNA purification assays according to the instruction manual of the Chromatin Isolation by RNA Purification (ChIRP) Kit (Bes5104, BersinBio, Guangzhou, China). Briefly, 4 × 107 HPAECs were collected and crosslinked with 1% formaldehyde for 20 min at room temperature. Crosslinking was stopped by adding glycine to the cell suspension for 5 min. Then, the cells were lysed with CHIRP lysis buffer and sonicated to obtain DNA fragments of approximately 100–500 bp. Samples were precleared and incubated with FENDRR probes (TFO1, TFO2) at 37 °C for 180 min. Finally, DNA was isolated and subjected to qPCR. The specific biotinylated probes TFO1 and TFO2 were synthesized by GenePharma (Shanghai, China). The sequences are shown in Table 1.

Dual-luciferase reporter assay

The DRP1 promoter fragment containing the FENDRR binding site was cloned into the GV238 plasmid expressing luciferase (Genepharma, Shanghai, China). HPAECs were cotransfected with the FENDRR expression plasmid and DRP1 plasmid expressing luciferase with Lipofectamine 2000 for 48 h. Then, the luciferase activities were measured by the dual-luciferase reporter assay system (Promega, USA).

Electrophoretic mobility shift assay (EMSA)

The specific biotinylated probe containing the DRP1 TSS fragment and the transcribed FENDRR TFO fragment was synthesized by GenePharma (Shanghai, China). The electrophoretic mobility shift assay (EMSA) was performed by a Chemiluminescent EMSA Kit (GS009, Beyotime, Shanghai, China) following the manufacturer’s instructions. In brief, the biotinylated DRP1 TSS and the synthesized FENDRR TFO2 were reacted in 10 mL of binding reaction buffer at room temperature for 20 min. Then, the sample was added to a 4% nondenatured polyacrylamide gel for electrophoresis purposes and transferred onto a nylon membrane, followed by UV crosslinking at 245 wavelengths. The membrane was incubated with streptavidin-HRP conjugate and enhanced chemiluminescent reagent imaging.

Methylation-specific PCR (MSP)

The methylation level of the DRP1 promoter region was detected by a GENMED Universal Gene Methylation Detection Kit (GENMED Scientifics INC.USA). In brief, a genomic DNA extraction kit (K0512, Thermo Scientific, USA) was used to extract genomic DNA. Then, 2 µg of DNA was transformed with GENMED reagents and subsequently subjected to PCR. The PCR programs were as follows: predenaturation at 95 °C for 2 min, 35 cycles of denaturation at 95 °C for 30 s, 57 °C for 90 s, and annealing at 72 °C for 30 s, with the last extension at 72 °C for 5 min. Finally, the PCR products were analyzed by 2% agarose gel electrophoresis and captured with a gel imaging system. The primers for methylation and unmethylation of the DRP1 promoter were shown in Table 1.

Global RNA m6A quantification

The EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric, P-9005, Epigentek Group Inc., Farmingdale, NY) was used to detect global m6A modifications in total HPAEC RNAs following the manufacturer’s instructions. Briefly, 2 µL of NC, 2 µL of PC and 200 ng of RNA were added into strip wells, and the solution was mixed. m6A were detected using capture and detection antibodies. The detected signal was enhanced and then quantified colorimetrically at a wavelength of 450 nm in a spectrophotometer.

m6A dot blot assay

Two micrograms of total RNA were deposited on a nylon membrane (FFN10, Beyotime Biotechnology, Shanghai, China), and then the nylon membrane was crosslinked by UV for 3 min. Next, the nylon membrane was stained by using methylene blue. Subsequently, the nylon membrane was blocked for 1 h in blocking buffer, and the membrane was incubated with m6A antibody (1 µg/mL, A-1801, Epigentek Group Inc., Farmingdale, NY) at 4 ℃ overnight. The membrane was incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h and enhanced chemiluminescent reagent imaging.

Flow cytometry assay

Pyroptosis of HPAECs was detected by using an Annexin V-FITC Detection Kit (C1062S, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. HPAECs were collected and stained with annexin V-FITC and PI at room temperature for 20 min. Afterward, the samples were analyzed using a BD FACSCalibur Flow Cytometer (BD Biosciences, Bedford, MA).

Correlation analysis

Total RNA samples were extracted from HPAECs of the normoxia group and hypoxia group, and reverse transcribed into cDNA. Then, real-time PCR was used to measure the mRNA expression levels of FENDRR and DRP1. The correlation of FENDRR and DRP1 was analyzed using the Pearson correlation test of GraphPad Prism 8.0, and P < 0.05 was considered significant.

Caspase-1 activity assay

The caspase-1 activity was detected using a caspase-1 activity assay kit (C1102, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, 2 × 106 HPAECs were harvested and lysed on ice for 15 min, centrifuged at 16,000×g for 15 min, and the supernatant was mixed with synthetic tetrapeptide Ac-YVAD-pNA and incubated at 37 °C for overnight. Finally, absorbance at 405 nm was recorded. The concentrations of total proteins were measured by a Bradford assay kit (P0006, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The caspase-1 activity was calculated by the standard curve of pNA.

Bioinformatic analysis

To analyze FENDRR localization, the lncATLAS website (http://lncatlas.crg.eu/) was used. Secondary structure analysis of FENDRR was performed using the RNAfold web server (http://rna.tbi.univie.ac.at/cgibin/RNAWebSuite/RNAfold.cgi). The target proteins prediction of FENDRR was performed through AnnoLnc (http://annolnc.gao-lab.org/index.php) and RNAInter (https://www.rna-society.org/raid/). A Venn diagram was shown using Venny2.1 web server (https://bioinfogp.cnb.csic.es/tools/venny/index.html). KEGG and GO enrichment were analyzed with the DAVID website (https://david.ncifcrf.gov/). The FENDRR TFO sequence and DRP1 promoter TTS sequence were identified with LongTarget (http://lncrna.smu.edu.cn/show/DNATriplex). Space structure docking of FENDRR TFO2 and DRP1 TTS was performed using HNADOCK Server (http://huanglab.phys.hust.edu.cn/hnadock/). CpG islands in the DRP1 gene promoter region were analyzed using Methyl Primer Express (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi). The N6-methyladenosine (m6A) modification site of FENDRR was predicted using the SRAMP prediction server (http://www.cuilab.cn/sramp).

Statistical analysis

Statistical analyses were performed using GraphPad Prism Software 8.0 (GraphPad Software Inc.). Data are expressed as mean ± SD. All expression values were checked for normal distribution before statistical. Student's t test was used to compare the data between two groups and one-way ANOVA with Tukey post hoc test was used to compare between multiple groups. For non-normally distributed data, we performed nonparametric analyses such as the Mann–Whitney U test for two groups or Kruskal–Wallis test followed by Dunn post-test for multiple groups. Results with 2-tailed of P < 0.05 were considered statistically significant.