{"paper_id":"132642e7-6726-4214-bc36-ffe3e64d2d89","body_text":"LncRNAH19 acts as a ceRNA of let-7g to facilitate EndMT in hypoxic pulmonary hypertension via regulating TGF-β signalling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article LncRNAH19 acts as a ceRNA of let-7g to facilitate EndMT in hypoxic pulmonary hypertension via regulating TGF-β signalling pathway xin Yu, Jiabing Huang, Xu Liu, Juan Li, Miao Yu, Minghui Li, Yuliang Xie, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4367962/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background Hypoxic pulmonary hypertension (HPH) is a challenging lung arterial disorder with remarkably high incidence and mortality rates, and the efficiency of current HPH treatment strategies is unsatisfactory. Endothelial-to-mesenchymal transition (EndMT) in the pulmonary artery plays a crucial role in HPH. Previous studies have shown that lncRNA-H19 (H19) is involved in many cardiovascular diseases by regulating cell proliferation and differentiation but the role of H19 in EndMT in HPH has not been defined. Methods In this research, the expression of H19 was investigated in PAH human patients and rat models. Then, we established a hypoxia-induced HPH rat model to evaluate H19 function in HPH by Echocardiography and hemodynamic measurements. Moreover, luciferase reporter gene detection, and western blotting were used to explore the mechanism of H19. Results Here, we first found that the expression of H19 was significantly increased in the endodermis of pulmonary arteries and that H19 deficiency obviously ameliorated pulmonary vascular remodelling and right heart failure in HPH rats, and these effects were associated with inhibition of EndMT. Moreover, an analysis of luciferase activity indicated that microRNA-let-7g (let-7g) was a direct target of H19. H19 deficiency or let-7g overexpression can markedly downregulate the expression of TGFβR1, a novel target gene of let-7g. Furthermore, inhibition of TGFβR1 induced similar effects to H19 deficiency. Conclusions In summary, our findings demonstrate that the H19/let-7g/TGFβR1 axis is crucial in the pathogenesis of HPH by stimulating EndMT. Our study may provide new ideas for further research on HPH therapy in the near future. hypoxic pulmonary hypertension lncRNA-H19 endothelial-to-mesenchymal transition microRNA-let-7 g TGFβR1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Pulmonary hypertension (PH) is a refractory pulmonary vascular remodelling disease. 1 Hypoxia has been identified as a high-risk factor inducing the development of PH. 2 Clinically, hypoxic pulmonary hypertension (HPH) has been classified as the third category of this disease, which is common in individuals with chronic lung disease or living at high altitudes. 3 Although a growing armamentarium of available therapeutics, such as vasodilators, anticoagulants and diuretics, has significantly improved the management of the disease in the past decade, few cases of HPH have been eradicated. 4 , 5 The 3-year survival rate of HPH patients is significantly worse than that of patients with other diseases. 6 Pulmonary artery remodelling, which results from an excessive increase in pulmonary artery smooth muscle cells (PASMCs), is a key event of HPH, leading to angio-obliterative vascular structural changes and excessive vasoconstriction. 7 , 8 The increased in PASMCs can be derived from resident PASMCs itself, epithelial cells, fibroblasts and pericytes. 9 Recently, pulmonary artery endothelial cells (PAECs) were shown to contribute to vascular remodelling in HPH though their transformation into mesenchymal or SM-like phenotype cells, which called endothelial-to-mesenchymal transition (EndMT), that then migrate into their underlying tissues. 10 11 Other studies also shown that inhibition of EndMT can attenuate pulmonary vascular remodelling and reduce pulmonary artery pressure. 12 Therefore, EndMT would be a promising therapeutic target for HPH treatment. Long noncoding RNAs (lncRNAs), a class of noncoding RNAs containing more than 200 nucleotides, participate in various biological activities by functioning as competing endogenous RNAs (ceRNAs) that compete for microRNA (miRNA) binding, thereby controlling the stability or translation of mRNAs targeted by miRNAs and altering their response to various stimuli at the transcriptional and posttranscriptional levels. 13 LncRNA-H19 (H19) is an imprinted gene located on chromosome 11 that is barely detectable in healthy adult animals but prominently expressed in endothelial cells after blood vessel injury. 14 , 15 Previous studies have shown that H19 is closely related to many cardiovascular diseases, such as myocardial ischaemia, heart failure and atherosclerosis. 16 , 17 Notably, the latest studies have proven that the H19 level was significantly increased in the blood of patients with end-stage idiopathic PAH and positively correlated with the degree of right ventricular hypertrophy. 18 However, whether H19 is involved in the pathological progression of HPH and its potential function remain largely unclear. Thus, we wanted to explore whether H19 was necessary for EndMT in HPH. In the present study, we investigated the role of H19 in pulmonary artery remodelling and EndMT in H19-deficient rat HPH model. We further explored the underling molecular mechanisms of H19 function during the EndMT in both primary rat PAECs and human PAECs under hypoxic conditions. Materials and methods 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 GSE24988 20 , GSE113439 21 and GSE117261 22 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 S1 A-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% O 2 , 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% H 2 O 2 , 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% CO 2 . 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% O 2 ) 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. Results H19 was increased in the lung tissue of PH patients according to DEG analysis We performed DEG analysis which method similar to that reported in an earlier study we published. In this paper, we analysed 135 PH and 58 normal samples of lung tissue after combining the GSE24988, GSE113439 and GSE117261 datasets (Fig. 1 A-B). The limma program was used to compare the DEGs between the two groups and found 43 DEGs. These DEGs may be able to differentiate between PH and normal patients, according to heatmaps created using hierarchical cluster analysis (Fig. 1 C). We used R to analyze the GSE24988, GSE113439, and GSE117261 datasets, and volcano plots were used to show the differences between PH and normal tissues (Fig. 1 D). In the two sets of differentially expressed genes, we discovered 26 upregulated and 17 downregulated genes (Fig. 1 E). According to the GEO database, we observed that the expression of H19 was noticeably higher in the lung tissue of PH patients (Fig. 1 F). H19 was increased in HPH rats and hypoxia-treated PAECs To determine the expression patterns of H19 in the development of HPH, rats were exposed to a hypoxic environment (10% O 2 ) for 28 days. In comparison to the control group, HPH rats had considerably greater RVSP and mPAP (Fig. 2 A-B). Additionally, when compared with the control group, HPH rats had a far greater RV/LV + S ratio (Fig. 2 C). The thickness of the pulmonary vascular wall and collagen fibrosis in the perivasculature were substantially more severe in the HPH group than in the control group, according to the results of HE and Masson staining (Fig. 2 D). Previous research has demonstrated a tight connection between EndMT and pulmonary vascular remodelling in HPH patients. The expression of endothelium markers (CD31 or vWF) and mesothelial markers (α-SMA or vimentin) was detected using double-labelling immunofluorescence. As presented in Fig. 2 E, the fluorescence intensity of CD31 or vWF was significantly suppressed and that of α-SMA or vimentin was increased in HPH compared with controls. Moreover, the qRT‒PCR results (Fig. 2 F) showed that H19 expression was remarkably greater in the pulmonary arteries of HPH rats than it was in the control group. The in situ hybridization outcomes (Fig. 2 G) showed that only a few cells in the lung tissue sections of the control group were stained with H19-specific probes. In contrast, the hypoxic group's lung tissue had a considerably larger H19-positive region. In addition, the results of in vitro experiments showed that hypoxia (3%) treatment increased H19 levels in primary RPAECs and HPAECs (Fig. 2 H). H19 deficiency reduced RVSP and improved RV function in HPH rats To further elucidate the relationship between H19 and HPH, we subjected H19-deficient rats (H19 −/− ) to hypoxia for 28 days. The genomic region of the rat H19 locus is diagrammed in Fig. S1 (A) . PCR Fig. S1 (B) and in situ hybridization Fig. S1 (C) for H19 were confirmed that H19 was successfully knocked out in SD rats. The RVSP (Fig. 3 A-B), the ratio of RV/WT (Fig. 3 C), RV/LV + S (Fig. 3 D) and RV/TL (Fig. 3 E) ratios of H19 −/− -HPH rats considerably lower than those of the WT-HPH group. Furthermore, the WT-HPH rat exhibited a dramatic increase in TAPSE, PAAT and PAAT/PAET when compared with the control group, according to the results of echocardiographic Doppler and M-mode tracing (Fig. 3 F-K). In contrast, the indices of RV function in the H19 −/− -HPH group were similar to those in the control group. These results suggest that H19 gene deficiency can protect rats against hypoxia-induced pulmonary hypertension and RV dysfunction. H19 deficiency improved pulmonary vascular remodelling in HPH rats Vascular lumen stenosis caused by pulmonary artery remodelling is the root cause of increased pulmonary artery pressure and right heart failure in patients with HPH. As shown in Fig. 4 A, pulmonary arteries obtained from WT-HPH group rats exhibited a ruptured elastic membrane, which was remarkably reversed by H19 deficiency. The thickness of the pulmonary vascular wall and collagen fibrosis in the perivasculature were far higher in the HPH group than in the control group, and both characteristics were obviously diminished by H19 absence, according to the results of HE and VG staining ( Fig. 4 B-C ). The results of histomorphometric analysis further confirmed this finding (Fig. 4 D-E). In conclusion, these results suggest that H19 gene knockout can improve pulmonary vascular remodelling induced by HPH in rats. H19 deficiency inhibited EndMT in HPH Using double-labelling immunofluorescence to identify the expression of CD31 and -SMA, the impact of H19 on EndMT in HPH rats was investigated. As presented in Fig. 5 A, the fluorescence intensity of CD31 was significantly suppressed and that of α-SMA was increased in WT-HPH cells compared with controls. However, there was no discernible difference between the H19-/-HPH group rats and the control group in the CD31 and -SMA fluorescence intensities. Furthermore, we also investigated the role of H19 in EndMT in primary RPAECs (Cell identification results were shown in Fig. S2 ) and HPAECs and found that H19 knockdown significantly reduced hypoxia-induced EndMT in both cells. (Fig. 5 B-C). These results suggest that H19 deficiency-improved pulmonary artery remodelling is associated with inhibiting EndMT. H19 deficiency regulated EndMT of PAECs via the let-7 g/TGFβR1 axis Let-7 g was previously discovered to be downregulated in the pulmonary arteries of HPH rats and inhibited hypoxia-induced proliferation of PASMCs 7 , 23 . Interestingly, we subsequently found through bioinformatics that let-7 g may also be closely related to EndMT at PH. 24 In this study, we discovered that PAECs exposed to hypoxia displayed a clearly decreased expression of let-7 g (Fig. 6 A). The let-7 g mimic significantly inhibited EndMT in RPAECs (Fig. 6 B) and HPAECs ( Fig.S3 ), but the let-7 g inhibitor directly stimulated EndMT (Fig. 6 C). In terms of mechanism, TGFβR1 is a key protein in the TGFβ/Smad signalling pathway and plays an important role in EndMT. By using a luciferase reporter assay (Fig. 6 D), we were able to further confirm that let-7 g may bind to the TGF-R1 3′UTR and negatively control the production of TGF-R1 in PAECs (Fig. 6 E-F). In addition, knockdown of endogenous TGFβR1 with TGFβR1-siRNA efficaciously decreased hypoxia-induced Vimentin and α-SMA expression in HPAECs and restored CD31 protein levels (Fig. 6 G). These results demonstrated that let-7 g/TGFβ signalling is important for EndMT in HPH. According to recent research, lncRNAs can act as naturally occurring competitive RNAs (ceRNAs) to scavenge miRNAs that bind to the 3′UTR of mRNAs for their degradation. We found that H19 could target binding with let-7 g by bioinformatics websites (RegRNA 2.0) (Fig. 7 A) and verified by the luciferase reporter assay (Fig. 7 B). In addition, we found that H19 deficiency led to a considerable decrease in the amount of TGF-R1 expression that is elevated by hypoxia in both HPAECs (Fig. 7 C) and lung tissue (Fig. 7 D). These results demonstrated that H19 may regulate EndMT in PAECs through the let-7 g/TGFβR1 signalling axis. Discussion A gradual rise in pulmonary artery pressure and thickening of the pulmonary artery wall are two features of the complicated cardiovascular disease HPH. 25 Several lncRNAs have been shown to have a role in HPH pathogenesis in recent years. 26 , 27 In this study, we discovered that H19 was both increased by hypoxia in vitro and abundantly expressed in HPH rodent lungs. Moreover, H19 deficiency in vivo can effectively prevent pulmonary artery remodelling and right heart failure induced by hypoxia. Mechanistically, we found that TGFβR1 is a novel target of let-7 g and that H19 upregulated TGFβR1 expression by sponging let-7 g following hypoxia stimulation. Moreover, both in vivo and in vitro, H19 absence prevented EndMT in HPH cells. Vascular remodelling in PH is caused by excessive pulmonary artery smooth muscle cell proliferation and abnormal arterial endothelial cells. 28 A previous study revealed that H19 was highly expressed in monocrotaline-induced PAH and promoted the proliferation of PASMCs. 29 EndMT, a phenotypic change in endothelial cells, is increasingly becoming recognized as the crucial cytopathological basis of HPH in research. 30 , 31 Under hypoxic stimulation, PAECs can lose their original structural characteristics and phenotypes and then transdifferentiate into smooth muscle-like cells, which have high proliferative activity and contribute to the remodelling process of the pulmonary artery. 11 , 32 Our study investigated the role of H19 in HPH for the first time and found that H19 plays an important role during the conversion of PAECs into smooth muscle-like cells of under HPH. In the current experiment, we found that hypoxia treatment for 4 weeks can significantly promote pulmonary artery remodelling and EndMT in rats, and H19 knockout can significantly improve pathological changes. As a result, we propose that H19 may be a key player in the proliferation of PASMCs, EndMT, and right myocardial hypertrophy, making it an appropriate therapeutic target for PH. However, a recent study indicated that knockdown of H19 significantly promoted the proliferation of rPASMCs, which seems to contradict our finding. Different blood samples, different models, tissues and cell samples may have caused some discrepancies. 33 H19 with a primary structure up to 2.3 KB can act as a ceRNA to adsorb miRNA, thereby weakening its interference with the targeted mRNA. 29 , 34 Our findings imply that H19 sponges let-7 g, which is consistent with a prior publication. 35 Let-7 g is a member of the let-7 family, whose role in angiogenesis, vascular remodelling and epithelial mesenchymal transformation has been well documented. 36 , 37 In addition, let-7g was found to be downregulated in remodelled pulmonary arteries of HPH rats, whereas restoring let-7g could markedly blunt hypoxia-induced cell proliferation in PASMCs. 7 , 23 We subsequently found through bioinformatics that let-7g may also be closely related to EndMT at PH 24 and verified in this study. Other members of the Let-7 family, besides let-7 g, also take part in PH. Let-7b inhibited PASMC growth in monocrotaline-induced PAH, according to a prior study. 29 Moreover, the increased expression of ET-1 caused by the decreased let-7b expression influenced the proliferation of PASMC and PAEC in chronic thromboembolic PH. 38 It is well established that the TGF signalling pathway is essential for the development of HPH and vascular remodeling. 39 , 40 Our results were the first to confirm that TGFβR1, a key protein in the TGFβ signalling pathway, is a new target of let-7 g and that the H19-let-7 g-TGFβR1 axis, which stimulates EndMT, plays a role in the pathophysiology of HPH. This study does have certain restrictions, though. First off, while discovering by DEG analysis that H19 expression increased in the lung tissue of PH patients, we did not investigate H19 expression in the serum and lung tissue of HPH patients. Could blood H19 be used to diagnose or predict outcomes in PH patients? Second, it is not quite obvious how precisely hypoxia controls H19. H19 was strongly expressed in the 1-day-old rat aorta but was not found in the adult aorta, as was previously reported. H19 transcripts were hardly present in the carotid artery before injury, but they were plentiful at 7 and 14 days later and were largely localized to the neointima by in situ hybridization. 41 Furthermore, hypoxia has been shown to promote carcinogenic effects in glioblastoma by directly and indirectly inducing H19 expression through hypoxia-inducible transcription factor 1 alpha (HIF-1α ) activity. 42 There is a functional link between HIF-1α and H19 that determines H19 elevation in hypoxic cancer cells. 43 HIF-1, one of the hypoxia-responsive factors, is the main regulator of oxygen homeostasis and hypoxic adaptation in the lung. PASMC growth, EndMT development, and pulmonary vascular remodelling are all influenced by aberrant HIF-1 activation during PH etiology. 44 , 45 Consequently, we propose that the overexpression of H19 may be caused by both abnormal HIF-1 activity and injured pulmonary small arteries in PH. But more evidence is required to support the hypothesis. In summary, this work demonstrates that the expression of H19 was increased in the pulmonary arteries of HPH rats and hypoxic PAECs. In HPH rats, H19 absence alleviated right ventricular dysfunction and pulmonary vascular remodelling, which were both related to EndMT suppression via let-7g/TGFβ signalling (Fig. 8 ). Our data imply that H19 could provide a novel treatment target for HPH. Abbreviations PASMCs pulmonary artery smooth muscle cells HPH hypoxia-induced pulmonary hypertension EndMT endothelial-to-mesenchymal transition PAECs pulmonary artery endothelial cells. Declarations Funding This project was supported by funding from the National Natural Science Foundation of China (grant. no. 81960015), the Science Foundation for distinguished Young Scholars of Jiangxi Province (grant no. 20212ACB216008), the Young Talents Project Foundation from Science and Technology Department of Jiangxi Province (grant no. 20204BCJ23020) and the National College Students Innovation Training Program of Henan Province (grant no. 202310472003). Declaration of Generative AI and AI-assisted technologies in the writing Process AI-assisted technology was not used in the preparation of the work. Competing interests The authors have declared no competing interests exists. Author contributions Xin Yu : investigation (lead); methodology (equal); project administration (supporting); writing-original draft (lead). Jiabing Huang : investigation (supporting); methodology (equal); project administration (supporting); writing-original draft (equal). Xu Liu : writing-original draft (equal); investigation (supporting); methodology (equal); project administration (supporting). Juan Li: investigation (supporting); methodology (equal); visualization (equal). Miao Yu ： Funding acquisition (supporting); investigation (supporting); methodology (equal); visualization (equal); Minghui Li and Yuliang Xie: Investigation (supporting); methodology (supporting); visualization (equal). Ye Li and Junyu Qiu: methodology (supporting); visualization (supporting); writing – review & editing (Supporting). Zhou Xu: methodology (supporting); visualization (supporting). Tiantian Zhu: Funding acquisition (equal); project administration (equal); writing-review & editing (equal). Weifang Zhang: conceptualization (equal); Funding acquisition (lead); project administration (equal); writing-review & editing (lead). Data availability statement The authors confirm that the data supporting the findings of this study are available within the article ORCID Weifang Zhang https://orcid.org/0000-0002-8421-1395 Supplemental Material Fig. S1–S3 References Vazquez ZGS, Klinger JR. Guidelines for the Treatment of Pulmonary Arterial Hypertension. Lung. 2020;198(4):581–96. Welch CL, Chung WK. Channelopathy Genes in Pulmonary Arterial Hypertension. Biomolecules. 2022;12(2):265. Ruaro B, Baratella E, Caforio G, Confalonieri P, Wade B, Marrocchio C, Geri P, Pozzan R, Andrisano AG, Cova MA, Cortale M, Confalonieri M, Salton F. Chronic Thromboembolic Pulmonary Hypertension: An Update. Diagnostics (Basel) 2022, 12 (2). Tzoumas A, Peppas S, Sagris M, Papanastasiou CA, Barakakis PA, Bakoyiannis C, Taleb A, Kokkinidis DG, Giannakoulas G. Advances in treatment of chronic thromboembolic pulmonary hypertension. Thromb Res. 2022;212:30–7. Mandras SA, Mehta HS, Vaidya A. 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Supplementary Files Originalimageforchecking.docx FigureSupplemental1.docx FigureSupplemental2.docx FigureSupplemental3.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Jun, 2024 Reviews received at journal 07 Jun, 2024 Reviewers agreed at journal 30 May, 2024 Reviewers invited by journal 13 May, 2024 Editor assigned by journal 07 May, 2024 Submission checks completed at journal 06 May, 2024 First submitted to journal 04 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4367962\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":301591497,\"identity\":\"016abbaa-fc58-4be8-9622-322886ffee39\",\"order_by\":0,\"name\":\"xin Yu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"the Second Affiliated Hospital of Nanchang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"xin\",\"middleName\":\"\",\"lastName\":\"Yu\",\"suffix\":\"\"},{\"id\":301591498,\"identity\":\"735ff1b7-a073-4613-af8e-0730ca1bc54c\",\"order_by\":1,\"name\":\"Jiabing 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10:25:08\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4367962/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4367962/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":56479135,\"identity\":\"aa241fab-dbd6-4e16-92f4-bbcad5490816\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:58\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":290612,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eH19 was increased in lung tissue of PH patients through differential expressed gene (DEG) analysis.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003ePCA before combined; \\u003cstrong\\u003e(B)\\u003c/strong\\u003e PCA after combined; \\u003cstrong\\u003e(C)\\u003c/strong\\u003e Volcano map of DEGs. Magenta dots represent genes with |logFC| \\u0026gt; 0.5 and adj.Pval \\u0026lt;0.05. The blue nodes on the left represent downregulated DEGs and red nodes on the right represent upregulated DEGs; the gray nodes represent genes with p-value \\u0026gt; 0.05; \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Heatmap of 43 DEGs. The diagram presents the result of a two-way hierarchical clustering of all the DEGs and samples. Each row in the heatmap represents a sample, and each column represents gene. The color scale at the right of the heatmap represents the raw Z-score ranging from blue (low expression) to red (high expression); \\u003cstrong\\u003e(E)\\u003c/strong\\u003e H19 expression in lung tissue of PH patients through DEG analysis.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/d29a9c69545e5b9d65bd88ab.png\"},{\"id\":56479134,\"identity\":\"24406c20-605d-4445-99a7-0350382a2dfb\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:58\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1288487,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eH19 was increased in HPH rats and hypoxia-treated PAECs.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A-B) \\u003c/strong\\u003eQuantification of RVSP and mPAP in rats; \\u003cstrong\\u003e(C) \\u003c/strong\\u003eThe ratio of right ventricle weight to left ventricle plus ventricular septum weight (RV/LV+S) was measured; \\u003cstrong\\u003e(D) \\u003c/strong\\u003eMorphological analysis of the pulmonary artery was performed using HE and Masson staining;\\u003cstrong\\u003e (E) \\u003c/strong\\u003eThe expression of endothelial markers(CD31 or vWf) and mesothelial markers (α-SMA or vimentin) was detected using double-labelling immunofluorescence ; \\u003cstrong\\u003e(F) \\u003c/strong\\u003eThe expression of H19 in pulmonary artery of rats was detected by qRT-PCR; \\u003cstrong\\u003e(G)\\u003c/strong\\u003e The expression of H19 in lung tissue of rats in each group was detected by in situ hybridization (brown).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/0bb600594e970d2f67bc7ae0.png\"},{\"id\":56479739,\"identity\":\"fa180ae6-bf35-4d72-85b6-28b1d0efa2d2\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 18:06:59\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":748115,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eH19 deficiency reduced RVSP and improved RV function in HPH rats.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003eOscillogram of right ventricular systolic pressure in rats; \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Quantification of RVSP in rats; \\u003cstrong\\u003e(C)\\u003c/strong\\u003eQuantification of the ratio of RV weight to left ventricular + ventricular septal weight (RV/LV+S) in rats; \\u003cstrong\\u003e(D)\\u003c/strong\\u003eQuantification of the ratio of RV weight to tibial length (RV/TL) in rats; \\u003cstrong\\u003e(E)\\u003c/strong\\u003e The systolic displacement of tricuspid valve (brown) in rats was detected by echocardiograms in M model; \\u003cstrong\\u003e(F)\\u003c/strong\\u003e Quantification of tricuspid annular plane systolic excursion;\\u003cstrong\\u003e (G) \\u003c/strong\\u003epulmonary artery blood flow acceleration time (red) and pulmonary artery ejection time (white) were measured by echocardiograms in Doppler model; \\u003cstrong\\u003e(H)\\u003c/strong\\u003e Quantification of pulmonary artery blood flow acceleration time; \\u003cstrong\\u003e(I)\\u003c/strong\\u003e Quantification of pulmonary artery blood flow ejection time; \\u003cstrong\\u003e(J)\\u003c/strong\\u003eQuantification of the ratio of acceleration time to ejection time. Data were presented as mean ± SEM (n=6).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/28730d5827bc2c117de632a8.png\"},{\"id\":56479140,\"identity\":\"1fec31a6-e1d5-4b77-893d-96084171e26e\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:59\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1074577,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eH19 deficiency improved pulmonary vascular remodelling in HPH rats.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003eTransmission electron microscopy (TEM) images of lung tissues; \\u003cstrong\\u003e(B)\\u003c/strong\\u003e HE staining in lung tissue;\\u003cstrong\\u003e (C) \\u003c/strong\\u003eVG staining in lung tissue;\\u003cstrong\\u003e (D)\\u003c/strong\\u003e Quantification of the ratio of pulmonary artery wall thickness to vessel diameter in rats; \\u003cstrong\\u003e(E)\\u003c/strong\\u003e Quantification of the fibrosis area.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/c5466b50f5ec2746c49af7f3.png\"},{\"id\":56479138,\"identity\":\"298d88b7-a5a7-408f-af4d-cd56e2857d3e\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:58\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":567812,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eH19 deficiency inhibited EndMT in HPH.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003eImmunofluorescence double staining of CD31 and α-SMA in lung tissues (CD31 (red), α-SMA (green), DAPI (blue)); \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Immunofluorescence double staining of CD31 and α-SMA in RPAECs; \\u003cstrong\\u003e(C)\\u003c/strong\\u003e Immunofluorescence double staining of CD31 and α-SMA in HPAECs ;Data were presented as mean ± SEM (n=6).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/fd130734162d2a469d70ad84.png\"},{\"id\":56479144,\"identity\":\"2c83c98c-097b-47ca-8073-5ab8ffbcbfff\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:59\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1015772,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eH19 deficiency regulated EndMT of PAECs via the let-7 g/TGFβ signalling.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003eThe expression level of let-7g in PAECs ;\\u003cstrong\\u003e(B)\\u003c/strong\\u003eImmunofluorescence double staining of CD31 and α-SMA in RPAECs treated with let-7g mimic; \\u003cstrong\\u003e(C)\\u003c/strong\\u003e Immunofluorescence double staining of CD31 and α-SMA in RPAECs treated with let-7g inhibitor; \\u003cstrong\\u003e(D)\\u003c/strong\\u003e the potential binding sites of let-7g to TGFβR1 predicted by Targetscan 7.0 and quantification of the fluorescence intensity in HEK293T cells; \\u003cstrong\\u003e(E-F) \\u003c/strong\\u003eAfter transfection of let-7g mimic or inhibitor, the expression level of TGFβR1 protein in HPAECs; \\u003cstrong\\u003e(G)\\u003c/strong\\u003e Transfection of after transfection of TGFβR1-Si, the expression levels of CD31, Vimentin and α-SMA were detected by Western blot.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/808948cc1f684145a9e40aaa.png\"},{\"id\":56479146,\"identity\":\"6bc8f470-0861-451d-9441-5e3f5da5e1f5\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:59\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":645602,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eH19 deficiency ameliorated EndMT was associated with miR-let-7g/TGFβ signalling.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003eThe potential binding targets of H19 to let-7g was predicted by RegRNA 2.0 software; \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Quantification of the fluorescence intensity in HEK293T cells; \\u003cstrong\\u003e(C)\\u003c/strong\\u003eAfter transfection with H19-Si, the expression level of TGFβR1 protein in HPAECs was detected by Western blot; \\u003cstrong\\u003e(D)\\u003c/strong\\u003eImmunofluorescence staining of TGFβR1 in lung tissues (TGFβR1 (red), DAPI (blue)). Data were presented as mean ± SEM (n=6).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/ffc80e2fb8c8337e19d27a91.png\"},{\"id\":56479143,\"identity\":\"b234899c-cef3-423b-b077-90248e00a19d\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:59\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":424347,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eScheme of the regulation of H19-let-7 g-TGFβR1 axis to EndMT in HPH.\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/d55237f9dc870611b27c7402.png\"},{\"id\":56480049,\"identity\":\"341bbdc9-5b07-44c1-a1f0-0fab2c5b31f3\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 18:15:02\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":7137609,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/11e0d6dd-e949-40c3-8f1c-0fa74cbb524c.pdf\"},{\"id\":56479137,\"identity\":\"b84c252b-a5c6-481f-90c0-51abc5cbbd05\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:58\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1136983,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Originalimageforchecking.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/ae9019b488a7bc8ddf1ca357.docx\"},{\"id\":56479139,\"identity\":\"2add3062-2ca4-46d8-8742-2703b13e3640\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:58\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":4861310,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"FigureSupplemental1.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/52aece95dd2cd966fcfa37bf.docx\"},{\"id\":56479145,\"identity\":\"b46aa2c2-cf35-4732-bd84-58fbdc99381a\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:59\",\"extension\":\"docx\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":4004296,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"FigureSupplemental2.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/3ed36cad9d943d58c43ea6b2.docx\"},{\"id\":56479142,\"identity\":\"69d330fc-d568-4100-ac32-784bb156af88\",\"added_by\":\"auto\",\"created_at\":\"2024-05-14 17:58:59\",\"extension\":\"docx\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":4400569,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"FigureSupplemental3.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4367962/v1/da82601995463abf57305dcf.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"LncRNAH19 acts as a ceRNA of let-7g to facilitate EndMT in hypoxic pulmonary hypertension via regulating TGF-β signalling pathway\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003ePulmonary hypertension (PH) is a refractory pulmonary vascular remodelling disease.\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e Hypoxia has been identified as a high-risk factor inducing the development of PH.\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e Clinically, hypoxic pulmonary hypertension (HPH) has been classified as the third category of this disease, which is common in individuals with chronic lung disease or living at high altitudes. \\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003eAlthough a growing armamentarium of available therapeutics, such as vasodilators, anticoagulants and diuretics, has significantly improved the management of the disease in the past decade, few cases of HPH have been eradicated.\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e The 3-year survival rate of HPH patients is significantly worse than that of patients with other diseases.\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003ePulmonary artery remodelling, which results from an excessive increase in pulmonary artery smooth muscle cells (PASMCs), is a key event of HPH, leading to angio-obliterative vascular structural changes and excessive vasoconstriction.\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e The increased in PASMCs can be derived from resident PASMCs itself, epithelial cells, fibroblasts and pericytes.\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e Recently, pulmonary artery endothelial cells (PAECs) were shown to contribute to vascular remodelling in HPH though their transformation into mesenchymal or SM-like phenotype cells, which called endothelial-to-mesenchymal transition (EndMT), that then migrate into their underlying tissues.\\u003csup\\u003e10 11\\u003c/sup\\u003e Other studies also shown that inhibition of EndMT can attenuate pulmonary vascular remodelling and reduce pulmonary artery pressure.\\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e Therefore, EndMT would be a promising therapeutic target for HPH treatment.\\u003c/p\\u003e \\u003cp\\u003eLong noncoding RNAs (lncRNAs), a class of noncoding RNAs containing more than 200 nucleotides, participate in various biological activities by functioning as competing endogenous RNAs (ceRNAs) that compete for microRNA (miRNA) binding, thereby controlling the stability or translation of mRNAs targeted by miRNAs and altering their response to various stimuli at the transcriptional and posttranscriptional levels. \\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003eLncRNA-H19 (H19) is an imprinted gene located on chromosome 11 that is barely detectable in healthy adult animals but prominently expressed in endothelial cells after blood vessel injury.\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e Previous studies have shown that H19 is closely related to many cardiovascular diseases, such as myocardial ischaemia, heart failure and atherosclerosis.\\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e Notably, the latest studies have proven that the H19 level was significantly increased in the blood of patients with end-stage idiopathic PAH and positively correlated with the degree of right ventricular hypertrophy.\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e However, whether H19 is involved in the pathological progression of HPH and its potential function remain largely unclear. Thus, we wanted to explore whether H19 was necessary for EndMT in HPH.\\u003c/p\\u003e \\u003cp\\u003eIn the present study, we investigated the role of H19 in pulmonary artery remodelling and EndMT in H19-deficient rat HPH model. We further explored the underling molecular mechanisms of H19 function during the EndMT in both primary rat PAECs and human PAECs under hypoxic conditions.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eData collection and analysis of differentially expressed genes\\u003c/h2\\u003e \\u003cp\\u003eThe method we used is similar to that reported in an earlier study we published. \\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e Briefly, the gene expression profiling datasets GSE24988\\u003csup\\u003e20\\u003c/sup\\u003e, GSE113439\\u003csup\\u003e21\\u003c/sup\\u003e and GSE117261\\u003csup\\u003e22\\u003c/sup\\u003e are based on GPL6244 ([HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version]) were downloaded from the GEO database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.ncbi.nlm.nih.gov/geo/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.ncbi.nlm.nih.gov/geo/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). 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 \\u0026ldquo;sva\\u0026rdquo; R package. We identified differentially expressed genes (DEGs) using the \\u0026ldquo;limma\\u0026rdquo; package in R. Values with P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 and |log2Fold change (logFC)| \\u0026gt;0.5 were considered statistically significant.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eHPH rat model\\u003c/h2\\u003e \\u003cp\\u003eH19-deficient rats (H19\\u0026minus;/\\u0026minus;) 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\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2\\u0026deg;C). After that, the tails of the offspring rats were isolated and genotyped by PCR (Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eA-B). Eight-week-old male homozygous rats and their wild-type (WT) littermates were used in the experiment.\\u003c/p\\u003e \\u003cp\\u003eTo induce HPH, rats were placed in an atmospheric hypoxia incubator (Changjin, Changsha) at 10% O\\u003csub\\u003e2\\u003c/sub\\u003e, 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.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEchocardiographic assessment\\u003c/h2\\u003e \\u003cp\\u003eAfter 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.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eRight ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) measurement\\u003c/h2\\u003e \\u003cp\\u003eThe 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.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSampling\\u003c/h2\\u003e \\u003cp\\u003eAfter 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\\u0026thinsp;+\\u0026thinsp;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.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTransmission electronic microscope (TEM)\\u003c/h2\\u003e \\u003cp\\u003eThe 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).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMorphological staining\\u003c/h2\\u003e \\u003cp\\u003eLung 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-\\u0026micro;m 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).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIn situ hybridization (ISH)\\u003c/h2\\u003e \\u003cp\\u003eAn in situ hybridization (ISH) assay was conducted using an RNA ISH Kit (GDP1061, Servicebio, Wuhan, China) according to the manufacturer\\u0026rsquo;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% H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e, 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\\u0026deg;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\\u0026rsquo;-GGGCTAGAGGCTTGGCTCCAGGATGATGT (ttt CATCATCAT ACATCATCAT) 30\\u0026thinsp;\\u0026minus;\\u0026thinsp;3\\u0026rsquo;, and the sequence of the secondary probe was 5\\u0026rsquo;-DIG-tt-ATGATGATGT ATGATGATGT-3\\u0026rsquo;.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eHypoxia-induced HPH in PAECs\\u003c/h2\\u003e \\u003cp\\u003ePrimary 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\\u0026ndash;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\\u0026deg;C in the presence of 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. 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.\\u003c/p\\u003e \\u003cp\\u003ehuman 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\\u0026deg;C.\\u003c/p\\u003e \\u003cp\\u003eTo 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% O\\u003csub\\u003e2\\u003c/sub\\u003e) for 48 h.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell transfection\\u003c/h2\\u003e \\u003cp\\u003eSmall 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.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunofluorescence\\u003c/h2\\u003e \\u003cp\\u003eAfter 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\\u0026deg;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).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDual luciferase reporter assay\\u003c/h2\\u003e \\u003cp\\u003eThe 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\\u0026trade; luciferase reporter vector (Ribobio), generating corresponding constructs H19-WT and H19-MUT. Similarly, the 3\\u0026prime;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\\u0026trade; 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\\u0026trade; 3000. After 48 h, luciferase activity analysis was performed using an ONE-Glo\\u0026trade; EX Luciferase Assay System (E8110, Promega, USA), with Renilla luciferase activity as a control.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eqRT‒PCR\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA from pulmonary arteries or PACEs was extracted by using TRIzol (Invitrogen, ON, Canada). Then, the cDNA for PCR was produced using 2 \\u0026micro;g total RNA and KEIris RT mix with dsDNase (All-in-One) (Codonx, Beijing, China). Next, RNA expression levels were evaluated by using TB Green\\u0026reg; Premix Ex Taq\\u0026trade; 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:\\u003c/p\\u003e \\u003cp\\u003eh-H19-Forwards: 5\\u0026rsquo;-CGTGACAAGCAGGACATGACA-3\\u0026rsquo;\\u003c/p\\u003e \\u003cp\\u003eh-H19-Reverse: 5\\u0026rsquo;-CCATAGTGTGCCGACTCCG-3\\u0026rsquo;\\u003c/p\\u003e \\u003cp\\u003er-H19-Forwards: 5\\u0026rsquo;-CTAAGTCGATTGCACTGGTTTGG-3\\u0026rsquo;\\u003c/p\\u003e \\u003cp\\u003er-H19-Reverse: 5\\u0026rsquo;-ACACCCAGTTGCCCTCAGAC-3\\u0026rsquo;\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern blot\\u003c/h2\\u003e \\u003cp\\u003eThe total protein content was extracted by precooled lysis buffer and quantified by bicinchoninic acid (BCA) protein assay kit (WB6501, New Cell \\u0026amp; Molecular Biotech LTD, Suzhou, China). Subsequently, equal samples containing 30 \\u0026micro;g 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 \\u0026amp; Molecular Biotech) for 15 min and then incubated overnight at 4\\u0026deg;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 \\u0026amp; Molecular Biotech).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eThe data are presented as the means\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;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). \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was deemed statistically significant.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eH19 was increased in the lung tissue of PH patients according to DEG analysis\\u003c/h2\\u003e \\u003cp\\u003eWe performed DEG analysis which method similar to that reported in an earlier study we published. In this paper, we analysed 135 PH and 58 normal samples of lung tissue after combining the GSE24988, GSE113439 and GSE117261 datasets (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA-B). The limma program was used to compare the DEGs between the two groups and found 43 DEGs. These DEGs may be able to differentiate between PH and normal patients, according to heatmaps created using hierarchical cluster analysis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). We used R to analyze the GSE24988, GSE113439, and GSE117261 datasets, and volcano plots were used to show the differences between PH and normal tissues (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). In the two sets of differentially expressed genes, we discovered 26 upregulated and 17 downregulated genes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE). According to the GEO database, we observed that the expression of H19 was noticeably higher in the lung tissue of PH patients (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eF).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eH19 was increased in HPH rats and hypoxia-treated PAECs\\u003c/h2\\u003e \\u003cp\\u003eTo determine the expression patterns of H19 in the development of HPH, rats were exposed to a hypoxic environment (10% O\\u003csub\\u003e2\\u003c/sub\\u003e) for 28 days. In comparison to the control group, HPH rats had considerably greater RVSP and mPAP (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA-B). Additionally, when compared with the control group, HPH rats had a far greater RV/LV\\u0026thinsp;+\\u0026thinsp;S ratio (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). The thickness of the pulmonary vascular wall and collagen fibrosis in the perivasculature were substantially more severe in the HPH group than in the control group, according to the results of HE and Masson staining (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). Previous research has demonstrated a tight connection between EndMT and pulmonary vascular remodelling in HPH patients. The expression of endothelium markers (CD31 or vWF) and mesothelial markers (α-SMA or vimentin) was detected using double-labelling immunofluorescence. As presented in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE, the fluorescence intensity of CD31 or vWF was significantly suppressed and that of α-SMA or vimentin was increased in HPH compared with controls. Moreover, the qRT‒PCR results (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eF) showed that H19 expression was remarkably greater in the pulmonary arteries of HPH rats than it was in the control group. The in situ hybridization outcomes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eG) showed that only a few cells in the lung tissue sections of the control group were stained with H19-specific probes. In contrast, the hypoxic group's lung tissue had a considerably larger H19-positive region. In addition, the results of in vitro experiments showed that hypoxia (3%) treatment increased H19 levels in primary RPAECs and HPAECs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eH).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eH19 deficiency reduced RVSP and improved RV function in HPH rats\\u003c/h2\\u003e \\u003cp\\u003eTo further elucidate the relationship between H19 and HPH, we subjected H19-deficient rats (H19\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e) to hypoxia for 28 days. The genomic region of the rat H19 locus is diagrammed in \\u003cb\\u003eFig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e(A)\\u003c/b\\u003e. PCR \\u003cb\\u003eFig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e(B)\\u003c/b\\u003e and in situ hybridization \\u003cb\\u003eFig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e(C)\\u003c/b\\u003e for H19 were confirmed that H19 was successfully knocked out in SD rats. The RVSP (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA-B), the ratio of RV/WT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC), RV/LV\\u0026thinsp;+\\u0026thinsp;S (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD) and RV/TL (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE) ratios of H19\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e-HPH rats considerably lower than those of the WT-HPH group. Furthermore, the WT-HPH rat exhibited a dramatic increase in TAPSE, PAAT and PAAT/PAET when compared with the control group, according to the results of echocardiographic Doppler and M-mode tracing (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF-K). In contrast, the indices of RV function in the H19\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e-HPH group were similar to those in the control group. These results suggest that H19 gene deficiency can protect rats against hypoxia-induced pulmonary hypertension and RV dysfunction.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eH19 deficiency improved pulmonary vascular remodelling in HPH rats\\u003c/h2\\u003e \\u003cp\\u003eVascular lumen stenosis caused by pulmonary artery remodelling is the root cause of increased pulmonary artery pressure and right heart failure in patients with HPH. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA, pulmonary arteries obtained from WT-HPH group rats exhibited a ruptured elastic membrane, which was remarkably reversed by H19 deficiency. The thickness of the pulmonary vascular wall and collagen fibrosis in the perivasculature were far higher in the HPH group than in the control group, and both characteristics were obviously diminished by H19 absence, according to the results of HE and VG staining \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB-C\\u003cb\\u003e).\\u003c/b\\u003e The results of histomorphometric analysis further confirmed this finding (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD-E). In conclusion, these results suggest that H19 gene knockout can improve pulmonary vascular remodelling induced by HPH in rats.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eH19 deficiency inhibited EndMT in HPH\\u003c/h2\\u003e \\u003cp\\u003eUsing double-labelling immunofluorescence to identify the expression of CD31 and -SMA, the impact of H19 on EndMT in HPH rats was investigated. As presented in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA, the fluorescence intensity of CD31 was significantly suppressed and that of α-SMA was increased in WT-HPH cells compared with controls. However, there was no discernible difference between the H19-/-HPH group rats and the control group in the CD31 and -SMA fluorescence intensities. Furthermore, we also investigated the role of H19 in EndMT in primary RPAECs (Cell identification results were shown in \\u003cb\\u003eFig. S2\\u003c/b\\u003e) and HPAECs and found that H19 knockdown significantly reduced hypoxia-induced EndMT in both cells. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB-C). These results suggest that H19 deficiency-improved pulmonary artery remodelling is associated with inhibiting EndMT.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eH19 deficiency regulated EndMT of PAECs via the let-7 g/TGFβR1 axis\\u003c/h2\\u003e \\u003cp\\u003eLet-7 g was previously discovered to be downregulated in the pulmonary arteries of HPH rats and inhibited hypoxia-induced proliferation of PASMCs\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. Interestingly, we subsequently found through bioinformatics that let-7 g may also be closely related to EndMT at PH.\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e In this study, we discovered that PAECs exposed to hypoxia displayed a clearly decreased expression of let-7 g (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA). The let-7 g mimic significantly inhibited EndMT in RPAECs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB) and HPAECs (\\u003cb\\u003eFig.S3\\u003c/b\\u003e), but the let-7 g inhibitor directly stimulated EndMT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eC). In terms of mechanism, TGFβR1 is a key protein in the TGFβ/Smad signalling pathway and plays an important role in EndMT. By using a luciferase reporter assay (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eD), we were able to further confirm that let-7 g may bind to the TGF-R1 3\\u0026prime;UTR and negatively control the production of TGF-R1 in PAECs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eE-F). In addition, knockdown of endogenous TGFβR1 with TGFβR1-siRNA efficaciously decreased hypoxia-induced Vimentin and α-SMA expression in HPAECs and restored CD31 protein levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eG). These results demonstrated that let-7 g/TGFβ signalling is important for EndMT in HPH.\\u003c/p\\u003e \\u003cp\\u003eAccording to recent research, lncRNAs can act as naturally occurring competitive RNAs (ceRNAs) to scavenge miRNAs that bind to the 3\\u0026prime;UTR of mRNAs for their degradation. We found that H19 could target binding with let-7 g by bioinformatics websites (RegRNA 2.0) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA) and verified by the luciferase reporter assay (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eB). In addition, we found that H19 deficiency led to a considerable decrease in the amount of TGF-R1 expression that is elevated by hypoxia in both HPAECs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eC) and lung tissue (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eD). These results demonstrated that H19 may regulate EndMT in PAECs through the let-7 g/TGFβR1 signalling axis.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eA gradual rise in pulmonary artery pressure and thickening of the pulmonary artery wall are two features of the complicated cardiovascular disease HPH.\\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e Several lncRNAs have been shown to have a role in HPH pathogenesis in recent years.\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e In this study, we discovered that H19 was both increased by hypoxia in vitro and abundantly expressed in HPH rodent lungs. Moreover, H19 deficiency in vivo can effectively prevent pulmonary artery remodelling and right heart failure induced by hypoxia. Mechanistically, we found that TGFβR1 is a novel target of let-7 g and that H19 upregulated TGFβR1 expression by sponging let-7 g following hypoxia stimulation. Moreover, both in vivo and in vitro, H19 absence prevented EndMT in HPH cells.\\u003c/p\\u003e \\u003cp\\u003eVascular remodelling in PH is caused by excessive pulmonary artery smooth muscle cell proliferation and abnormal arterial endothelial cells.\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e A previous study revealed that H19 was highly expressed in monocrotaline-induced PAH and promoted the proliferation of PASMCs.\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e EndMT, a phenotypic change in endothelial cells, is increasingly becoming recognized as the crucial cytopathological basis of HPH in research.\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e Under hypoxic stimulation, PAECs can lose their original structural characteristics and phenotypes and then transdifferentiate into smooth muscle-like cells, which have high proliferative activity and contribute to the remodelling process of the pulmonary artery.\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e Our study investigated the role of H19 in HPH for the first time and found that H19 plays an important role during the conversion of PAECs into smooth muscle-like cells of under HPH. In the current experiment, we found that hypoxia treatment for 4 weeks can significantly promote pulmonary artery remodelling and EndMT in rats, and H19 knockout can significantly improve pathological changes. As a result, we propose that H19 may be a key player in the proliferation of PASMCs, EndMT, and right myocardial hypertrophy, making it an appropriate therapeutic target for PH. However, a recent study indicated that knockdown of H19 significantly promoted the proliferation of rPASMCs, which seems to contradict our finding. Different blood samples, different models, tissues and cell samples may have caused some discrepancies.\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eH19 with a primary structure up to 2.3 KB can act as a ceRNA to adsorb miRNA, thereby weakening its interference with the targeted mRNA.\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e Our findings imply that H19 sponges let-7 g, which is consistent with a prior publication.\\u003csup\\u003e\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e Let-7 g is a member of the let-7 family, whose role in angiogenesis, vascular remodelling and epithelial mesenchymal transformation has been well documented.\\u003csup\\u003e\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e In addition, let-7g was found to be downregulated in remodelled pulmonary arteries of HPH rats, whereas restoring let-7g could markedly blunt hypoxia-induced cell proliferation in PASMCs.\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e We subsequently found through bioinformatics that let-7g may also be closely related to EndMT at PH\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e and verified in this study. Other members of the Let-7 family, besides let-7 g, also take part in PH. Let-7b inhibited PASMC growth in monocrotaline-induced PAH, according to a prior study.\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e Moreover, the increased expression of ET-1 caused by the decreased let-7b expression influenced the proliferation of PASMC and PAEC in chronic thromboembolic PH.\\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e It is well established that the TGF signalling pathway is essential for the development of HPH and vascular remodeling.\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e Our results were the first to confirm that TGFβR1, a key protein in the TGFβ signalling pathway, is a new target of let-7 g and that the H19-let-7 g-TGFβR1 axis, which stimulates EndMT, plays a role in the pathophysiology of HPH.\\u003c/p\\u003e \\u003cp\\u003eThis study does have certain restrictions, though. First off, while discovering by DEG analysis that H19 expression increased in the lung tissue of PH patients, we did not investigate H19 expression in the serum and lung tissue of HPH patients. Could blood H19 be used to diagnose or predict outcomes in PH patients? Second, it is not quite obvious how precisely hypoxia controls H19. H19 was strongly expressed in the 1-day-old rat aorta but was not found in the adult aorta, as was previously reported. H19 transcripts were hardly present in the carotid artery before injury, but they were plentiful at 7 and 14 days later and were largely localized to the neointima by in situ hybridization.\\u003csup\\u003e\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e Furthermore, hypoxia has been shown to promote carcinogenic effects in glioblastoma by directly and indirectly inducing H19 expression through hypoxia-inducible transcription factor 1 alpha (HIF-1α ) activity.\\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u003c/sup\\u003e There is a functional link between HIF-1α and H19 that determines H19 elevation in hypoxic cancer cells.\\u003csup\\u003e\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e\\u003c/sup\\u003e HIF-1, one of the hypoxia-responsive factors, is the main regulator of oxygen homeostasis and hypoxic adaptation in the lung. PASMC growth, EndMT development, and pulmonary vascular remodelling are all influenced by aberrant HIF-1 activation during PH etiology.\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e Consequently, we propose that the overexpression of H19 may be caused by both abnormal HIF-1 activity and injured pulmonary small arteries in PH. But more evidence is required to support the hypothesis.\\u003c/p\\u003e \\u003cp\\u003eIn summary, this work demonstrates that the expression of H19 was increased in the pulmonary arteries of HPH rats and hypoxic PAECs. In HPH rats, H19 absence alleviated right ventricular dysfunction and pulmonary vascular remodelling, which were both related to EndMT suppression via let-7g/TGFβ signalling (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e). Our data imply that H19 could provide a novel treatment target for HPH.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cdiv class=\\\"DefinitionList\\\"\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003ePASMCs\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003epulmonary artery smooth muscle cells\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eHPH\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003ehypoxia-induced pulmonary hypertension\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eEndMT\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eendothelial-to-mesenchymal transition\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003ePAECs\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003epulmonary artery endothelial cells.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis project was supported by funding from the National Natural Science Foundation of China (grant. no. 81960015), the\\u0026nbsp;Science Foundation for\\u0026nbsp;distinguished Young Scholars\\u0026nbsp;of\\u0026nbsp;Jiangxi Province\\u0026nbsp;(grant no.\\u0026nbsp;20212ACB216008), the Young Talents Project Foundation from Science and Technology Department of Jiangxi Province (grant no. 20204BCJ23020) and the National College Students Innovation Training Program of Henan Province (grant no. 202310472003).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDeclaration of Generative AI and AI-assisted technologies in the writing Process\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAI-assisted technology was not used in the preparation of the work.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors have declared no competing interests exists.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eXin Yu\\u003c/strong\\u003e: investigation (lead); methodology (equal); project administration (supporting); writing-original draft (lead). \\u003cstrong\\u003eJiabing Huang\\u003c/strong\\u003e: investigation (supporting); methodology (equal); project administration (supporting); writing-original draft (equal). \\u003cstrong\\u003eXu Liu\\u003c/strong\\u003e: writing-original draft (equal); investigation (supporting); methodology (equal); project administration (supporting). \\u003cstrong\\u003eJuan Li:\\u003c/strong\\u003e investigation (supporting); methodology (equal); visualization (equal). \\u003cstrong\\u003eMiao Yu\\u003c/strong\\u003e\\u003cstrong\\u003e：\\u003c/strong\\u003eFunding acquisition (supporting); investigation (supporting); methodology (equal); visualization (equal); \\u003cstrong\\u003eMinghui Li and Yuliang Xie:\\u003c/strong\\u003e Investigation (supporting); methodology (supporting); visualization (equal).\\u003cstrong\\u003e\\u0026nbsp;Ye Li and\\u003c/strong\\u003e \\u003cstrong\\u003eJunyu Qiu:\\u0026nbsp;\\u003c/strong\\u003emethodology (supporting); visualization (supporting); writing \\u0026ndash; review \\u0026amp; editing (Supporting). \\u003cstrong\\u003eZhou Xu:\\u003c/strong\\u003e methodology (supporting); visualization (supporting). \\u003cstrong\\u003eTiantian Zhu:\\u003c/strong\\u003e Funding acquisition (equal); project administration (equal); writing-review \\u0026amp; editing (equal). \\u003cstrong\\u003eWeifang Zhang:\\u003c/strong\\u003e conceptualization (equal); Funding acquisition (lead); project administration (equal); writing-review \\u0026amp; editing (lead).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability statement\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors confirm that the data supporting the findings of this study are available within the article\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eORCID\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eWeifang Zhang https://orcid.org/0000-0002-8421-1395\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSupplemental Material\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFig. S1\\u0026ndash;S3\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eVazquez ZGS, Klinger JR. 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Knockdown of LncRNA-H19 Ameliorates Kidney Fibrosis in Diabetic Mice by Suppressing miR-29a-Mediated EndMT. Front Pharmacol. 2020;11:586895.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi C, Li Y, Zhuang M, Zhu B, Zhang W, Yan H, Zhang P, Li D, Yang J, Sun Y, Cui Q, Chen H, Jin P, Xia Z, Sun Y. Long noncoding RNA H19 act as a competing endogenous RNA of Let-7g to facilitate IEC-6 cell migration and proliferation via regulating EGF. J Cell Physiol. 2021;236(4):2881\\u0026ndash;92.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHsu PY, Hsi E, Wang TM, Lin RT, Liao YC, Juo SH. MicroRNA let-7g possesses a therapeutic potential for peripheral artery disease. J Cell Mol Med. 2017;21(3):519\\u0026ndash;29.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang XH, Qian Y, Li Z, Zhang NN, Xie YJ. Let-7g-5p inhibits epithelial-mesenchymal transition consistent with reduction of glioma stem cell phenotypes by targeting VSIG4 in glioblastoma. Oncol Rep. 2016;36(5):2967\\u0026ndash;75.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGuo L, Yang Y, Liu J, Wang L, Li J, Wang Y, Liu Y, Gu S, Gan H, Cai J, Yuan JX, Wang J, Wang C. Differentially expressed plasma microRNAs and the potential regulatory function of Let-7b in chronic thromboembolic pulmonary hypertension. PLoS ONE 2014, 9 (6), e101055.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHiepen C, Jatzlau J, Hildebrandt S, Kampfrath B, Goktas M, Murgai A, Cuellar Camacho JL, Haag R, Ruppert C, Sengle G, Cavalcanti-Adam EA, Blank KG, Knaus P. BMPR2 acts as a gatekeeper to protect endothelial cells from increased TGFbeta responses and altered cell mechanics. PLoS Biol 2019, 17 (12), e3000557.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYuan S, Dong M, Zhang H, Jiang X, Yan C, Ye R, Zhou H, Chen L, Lian H, Jin W. Ginsenoside PPD inhibit the activation of HSCs by directly targeting TGFbetaR1. Int J Biol Macromol. 2022;194:556\\u0026ndash;62.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKim DK, Zhang L, Dzau VJ, Pratt RE. H19, a developmentally regulated gene, is reexpressed in rat vascular smooth muscle cells after injury. J Clin Invest. 1994;93(1):355\\u0026ndash;60.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWu W, Hu Q, Nie E, Yu T, Wu Y, Zhi T, Jiang K, Shen F, Wang Y, Zhang J, You Y. Hypoxia induces H19 expression through direct and indirect Hif-1alpha activity, promoting oncogenic effects in glioblastoma. Sci Rep. 2017;7:45029.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMatouk IJ, Mezan S, Mizrahi A, Ohana P, Abu-Lail R, Fellig Y, Degroot N, Galun E, Hochberg A. The oncofetal H19 RNA connection: hypoxia, p53 and cancer. Biochim Biophys Acta. 2010;1803(4):443\\u0026ndash;51.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWilkins MR, Ghofrani HA, Weissmann N, Aldashev A, Zhao L. Pathophysiology and treatment of high-altitude pulmonary vascular disease. Circulation. 2015;131(6):582\\u0026ndash;90.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSemenza GL. Hypoxia-inducible factors in physiology and medicine. Cell. 2012;148(3):399\\u0026ndash;408.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"respiratory-research\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"rere\",\"sideBox\":\"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)\",\"snPcode\":\"12931\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12931/3\",\"title\":\"Respiratory Research\",\"twitterHandle\":\"@RespiratoryBMC\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"hypoxic pulmonary hypertension, lncRNA-H19, endothelial-to-mesenchymal transition, microRNA-let-7 g, TGFβR1\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4367962/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4367962/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eHypoxic pulmonary hypertension (HPH) is a challenging lung arterial disorder with remarkably high incidence and mortality rates, and the efficiency of current HPH treatment strategies is unsatisfactory. Endothelial-to-mesenchymal transition (EndMT) in the pulmonary artery plays a crucial role in HPH. Previous studies have shown that lncRNA-H19 (H19) is involved in many cardiovascular diseases by regulating cell proliferation and differentiation but the role of H19 in EndMT in HPH has not been defined.\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e \\u003cp\\u003eIn this research, the expression of H19 was investigated in PAH human patients and rat models. Then, we established a hypoxia-induced HPH rat model to evaluate H19 function in HPH by Echocardiography and hemodynamic measurements. Moreover, luciferase reporter gene detection, and western blotting were used to explore the mechanism of H19.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eHere, we first found that the expression of H19 was significantly increased in the endodermis of pulmonary arteries and that H19 deficiency obviously ameliorated pulmonary vascular remodelling and right heart failure in HPH rats, and these effects were associated with inhibition of EndMT. Moreover, an analysis of luciferase activity indicated that microRNA-let-7g (let-7g) was a direct target of H19. H19 deficiency or let-7g overexpression can markedly downregulate the expression of TGFβR1, a novel target gene of let-7g. Furthermore, inhibition of TGFβR1 induced similar effects to H19 deficiency.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e \\u003cp\\u003eIn summary, our findings demonstrate that the H19/let-7g/TGFβR1 axis is crucial in the pathogenesis of HPH by stimulating EndMT. Our study may provide new ideas for further research on HPH therapy in the near future.\\u003c/p\\u003e\",\"manuscriptTitle\":\"LncRNAH19 acts as a ceRNA of let-7g to facilitate EndMT in hypoxic pulmonary hypertension via regulating TGF-β signalling pathway\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-05-14 17:58:53\",\"doi\":\"10.21203/rs.3.rs-4367962/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-06-25T10:02:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-06-07T12:27:43+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"174065131849137915351425326738009842250\",\"date\":\"2024-05-30T08:16:54+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-05-13T11:58:22+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-05-07T18:48:24+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-05-07T03:36:55+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Respiratory Research\",\"date\":\"2024-05-04T10:20:45+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"respiratory-research\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"rere\",\"sideBox\":\"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)\",\"snPcode\":\"12931\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12931/3\",\"title\":\"Respiratory Research\",\"twitterHandle\":\"@RespiratoryBMC\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"d2d4eb93-0d82-4778-9dae-a142d79d7e20\",\"owner\":[],\"postedDate\":\"May 14th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-06-28T14:08:29+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-05-14 17:58:53\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4367962\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4367962\",\"identity\":\"rs-4367962\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}