LncRNA PAXIP1‐AS1 fosters the pathogenesis of pulmonary arterial hypertension via ETS1/WIPF1/RhoA axis

Abstract Pulmonary arterial hypertension (PAH) is a life‐threatening disease featured with elevated pulmonary vascular resistance and progressive pulmonary vascular remodelling. It has been demonstrated that lncRNA PAXIP1‐AS1 could influence the transcriptome in PAH. However, the exact molecular mechanism of PAXIP1‐AS1 in PAH pathogenesis remains largely unknown. In this study, in vivo rat PAH model was established by monocrotaline (MCT) induction and hypoxia was used to induce in vitro PAH model using human pulmonary artery smooth muscle cells (hPASMCs). Histological examinations including H&E, Masson's trichrome staining and immunohistochemistry were subjected to evaluate the pathological changes of lung tissues. Expression patterns of PAXIP1‐AS1 and RhoA were assessed using qRT‐PCR and Western blotting, respectively. CCK‐8, BrdU assay and immunofluorescence of Ki67 were performed to measure the cell proliferation. Wound healing and transwell assays were employed to evaluate the capacity of cell migration. Dual‐luciferase reporter assay, co‐immunoprecipitation, RIP and CHIP assays were employed to verify the PAXIP1‐AS1/ETS1/WIPF1/RhoA regulatory network. It was found that the expression of PAXIP1‐AS1 and RhoA was remarkably higher in both lung tissues and serum of MCT‐induced PAH rats, as well as in hypoxia‐induced hPASMCs. PAXIP1‐AS1 knockdown remarkably suppressed hypoxia‐induced cell viability and migration of hPASMCs. PAXIP1‐AS1 positively regulated WIPF1 via recruiting transcriptional factor ETS1, of which knockdown reversed PAXIP1‐AS1‐mediated biological functions. Co‐immunoprecipitation validated the WIPF1/RhoA interaction. In vivo experiments further revealed the role of PAXIP1‐AS1 in PAH pathogenesis. In summary, lncRNA PAXIP1‐AS1 promoted cell viability and migration of hPASMCs via ETS1/WIPF1/RhoA, which might provide a potential therapeutic target for PAH treatment.


| INTRODUC TI ON
Patients with a resting mean pulmonary artery pressure of 25 mm Hg or above are diagnosed as pulmonary hypertension (PH), which is divided into five groups in clinic. 1,2 Pulmonary arterial hypertension (PAH) is one type of PH, featured with vasoconstriction obstructed pulmonary vasculature, adverse vascular remodelling, vascular fibrosis and stiffening structure. In the past decades, improved understanding of the pathophysiology of PAH leads to the development of efficient clinical drugs targeting vascular remodelling. [3][4][5][6][7][8][9] For example, the calcium channel blockers, drugs targeting nitric oxide pathway (inhaled NO, PDE5 inhibitors, soluble guanylate cyclase stimulators), endothelin receptor antagonists and drugs targeting the prostacyclin pathway have been reported to ameliorate the pathogenesis of PAH. [10][11][12][13][14] However, none of these agents is sufficient enough for PAH treatment. Thus, novel drug candidates generated from better understanding of the underlying mechanism of PAH are urgently needed.
In recent years, potential roles of long non-coding RNA (lncRNAs) have been reported in many diseases, including PAH. For instance, lncRNA TUG1 was found to promote vascular remodelling in PAH. 15 Zhou et al also showed that polymorphism of MALAT1 contributed to PAH susceptibility in Chinese population. 16 Moreover, Zahid et al further demonstrated that lncRNAs acted as both promoter and suppressor of pulmonary artery smooth muscle cells' (PASMCs) proliferation and migration. 17 Among these lncRNAs, PAXIP1-AS1 was recently reported to be up-regulated in PAH and might be associated with the hyperproliferative and migratory actions of IPAH smooth muscle cells. 18 However, deeper insights for how PAXIP1-AS1 participates in the pathogenesis of PAH are still urgently needed.
The normal human E26 transformation specific-1 (ETS1) is a widely accepted proto-oncogene, which has been found to promote cancer development including breast cancer, 19 ovarian cancer 20 and gastric cancer. 21 Recently, it has been verified that ETS1 also promoted cell proliferation and migration in glioma by regulation of PAXIP1-AS1. 22 However, whether such regulatory relationship exists in PAH remains largely unknown.
The small GTPase RhoA and its downstream effectors, the Rho kinases, are considered as important mediators in many cellular processes, such as differentiation, proliferation, survival and migration. Recently, accumulating evidence has presented that RhoA/ROCK pathway plays a vital role in the development and progression of PAH. For example, it has been identified that Rho kinase and RhoA activities were significantly increased in lung tissues of patients diagnosed with idiopathic PAH. 12 The activity of RhoA/ROCK pathway is also closely associated with PAH development. 23 Additionally, RhoA activation and ROCK levels were reported to be elevated in chronic hypoxic lungs. 24 Besides, in a recent research, RhoA was found as a downstream target of WIPF1, an oncogene in many cancers. 25 Interestingly, WIPF1 could be regulated by ETS1 in lung cancer. 26 However, the underlying mechanisms of relationship among ETS1, WIPF1 and RohA in PAH have not been noticed yet.
Thus, we aimed to investigate the role of PAXIP1-AS1 and its relationship with ETS1/WIPF1/RohA signalling in PAH and explore the downstream potential mechanisms participating in the regulation of PAH pathological process. Our study validated that PAXIP1-AS1 might be a therapeutic target for PAH treatment.

| Animal studies
A total of 60 adult male Sprague-Dawley rats (200 ± 20 g, 4-5 wk) obtained from Bioray Lab (Bioray, Shanghai, China) were kept in a pathogen-free environment with a 12-hr light/dark cycle with a temperature of 22 ± 1˚C and a humidity of 40%-60%. Animals were fed with a standard laboratory chow and water ad libitum for the duration of the experiments. The rats were randomly divided into normal group and PAH group. For induction of PAH, the rats were given by a subcutaneous injection of monocrotaline (MCT, Sigma Chemicals, St. Louis, MO, USA; 60 mg/kg), while the normal group was given a single injection of saline as a control. For treatment of PAH, animals injected with MCT were orally given fasudil (100 mg/kg per day) from the day given MCT injection. For inhibition of PAXIP1-AS1, tail vein injection was conducted with lentiviral plasmid that stably expressing sh-RNAs targeting PAXIP1-AS1 or negative control (lent-sh-PAXIP1-AS1/NC, active titre 2*10 8 TU/mL, GenePharma, Shanghai, China). The rats in all the groups were killed after intraperitoneal injection with 10% chloral hydrate, and samples of lung tissues were removed from each rat and were divided into two groups. One group was flash-frozen in liquid nitrogen and stored at -80˚C for Western blot analysis. The remaining tissues were fixed in 4% paraformaldehyde buffer for histopathological examinations. All animal care and experimental procedures were approved by the Animal Care Ethics and Use Committee of the Second Xiangya Hospital of Central South University and were performed in accordance with the guidelines of this Committee.

| Right ventricle system pressure (RVSP) measurements
After the animals were anaesthetized by inhalation of a mixture of isoflurane (4%) and oxygen, the polyethylene catheters were inserted into the right ventricle for haemodynamic measurements. A polygraph system (AP-601G, Nihon Kohden) was used to measure the right ventricle systolic pressure and systemic blood pressure.

| Histological examination
Paraffin-fixed lung tissues were cut into 4 μM sections using a rotary microtome (Leica, Mannheim, Germany) and were then subjected to HE staining. The orientation of collagen fibres was examined with Masson trichrome staining according to the manufacturer's instruction. For immunohistochemistry staining, slides were stained with antibodies against RhoA (ab54835, Abcam, Cambridge, USA; 1:100) and ɑ-SMA (ab124964, Abcam, Cambridge, USA; 1:100). The tissues were analysed using a microscope (DP73; Olympus Corporation, Tokyo, Japan).

| BrdU incorporation assay
The cell proliferation was determined using BrdU assay following the manufacturer's instructions. Briefly, the cells were grown on coverslips (Thermo Fisher Scientific) and then incubated with BrdU (20 μM) for 4 h. Cells were then permeabilized with 0.1% Triton X-100 in PBS and blocked with 3% FBS solution. Cellular DNA was denatured using DNasel treatment. The incorporated BrdU was stained with Alexa Fluor 647 anti-BrdU monoclonal antibody (BD Biosciences, USA). The nuclei were counter-stained with DAPI (Sigma). Images were acquired using a Carl Zeiss fluorescence microscope.

| Immunofluorescence of Ki67
For Ki67 staining, cells were fixed in ice-cold methanol for 15 min and allowed to dry for 20 min. Subsequently, cells were incubated with primary antibodies against Ki67 (Cell Signaling Technology, Danvers, MA, USA) at 4˚C overnight after being blocked, followed by a secondary antibody incubation. Nuclei were visualized by counterstaining the cells with DAPI (Invitrogen). Fluorescence intensity was recorded using a fluorescence microscope IX83 (Olympus).

| Wound healing assay
Cell migration ability of hPASMCs was tested using the scratch wound assay and then seeded into 6-well plates for 24 h. Cell layers were scratched using a 200 μl pipette tip to form wound gaps, and the cells were maintained in DMEM with 10% FBS. The cells were photographed at 0 and 24 h to record the wound width.

| Transwell assay of cell migration
The migratory capability was detected using transwell chambers (8 μm pores, Millipore, USA). Briefly, cell were digested, resuspended and then plated on the surface of upper chambers. The media containing 10% FBS was injected into the lower chambers to stimulate cell migration. After 8-h incubation, membranes at the subjacent sides of upper chambers were fixed with 4% PFA and then stained with 0.1% crystal violet stain solution (Solarbio, China). Images were taken by an optical microscope (Leica DM3000, Germany).

| Protein extraction and Western blotting
Protein was extracted using RIPA, and the concentration was de- Biotechnology (Heidelberg, Germany). Protein bands were then visualized using the enhanced chemiluminescence detection system (Bio-Rad Laboratories, Mississauga, Canada). The intensity of the bands was quantified using ImageJ software tools.

| RNA extraction, reverse transcription and quantitative PCR (qRT-PCR)
The expression of WIPF1, RhoA and ETS1 was determined by qRT- Primers used in this study are shown in Table 1.

| Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed using Pierce Agarose ChIP kit (Pierce) according to the manufacturer's instructions. Briefly, cells were transfected with sh-NC or sh-PAXIP1-AS1, respectively. Cells were cross-linked in 1% formaldehyde and lysed at 48 h post-transfection.
Chromatin was digested using micronuclease. Sheared DNA was incubated with anti-WIPF1 antibody. Normal IgG was used as a negative control. DNA was purified and analysed by PCR.

| Co-immunoprecipitation (Co-IP)
Cell extracts were incubated with antibodies against WIPF1 or RhoA (Cell Signaling Technologies, Boston, MA, USA) followed by addition of protein A/G-Sepharose overnight at 4˚C with constant head-totail rotations. Sepharose beads were pre-blocked with 2% BSA in PBS before being used for immunoprecipitation. After incubation, proteins bound to Sepharose beads were washed twice with the lysis buffer, four times with ice-cold PBS and mixed with 2x SDS-PAGE gel loading buffer and boiled for 5 min. Then, the supernatant (20 μl per lane) was loaded onto a gel for SDS-PAGE for electrophoresis and subsequent immunoblot was analysed with the specific antibodies.

| Luciferase reporter assay
The dual-luciferase reporter assay was utilized to determine the interaction of ETS1 and WIPF1 and was conducted according to the method described by Zhang et al. 28 Briefly, to obtain the mutant WIPF1 3'UTR

| Statistical analysis
The data were presented as mean ± standard deviation (SD) from three independent experiments. For comparisons between two groups, Student's t test was employed and comparisons between groups were analysed by using one-way ANOVA. Pearson analysis was used to analyse the correlation between factors. A two-sided value of P < .05 was considered statistically significant.

| Expression patterns of PAXIP1-AS1 and RhoA were increased in MCT-induced rat lung tissues
To investigate the role of PAXIP1-AS1 in PAH, in vivo rat PAH model was established by MCT induction. Firstly, RVSP in PAH rat was significantly elevated from 28 days after MCT injection ( Figure 1A).
Besides, as shown in Figure 1B higher in both lung tissues and serum of MCT-induced PAH rats than the control rats ( Figure 1D). Besides, the protein levels of RhoA were also significantly up-regulated in MCT-induced PAH model ( Figure 1E). Further, Pearson correlation analysis also revealed the positive correlation of RhoA and PAXIP1-AS1 in MCT-induced rat lung tissues (Figure 1F). Taken together, RhoA and PAXIP1-AS1 were increased in MCT-induced rat lung tissues, indicating their biological functions in PAH progression. All these results suggested that knockdown of PAXIP1-AS1 suppressed cell viability and migration of hPASMCs.

| PAXIP1-AS1 positively regulated the expression of WIPF1 through assembling ETS1
To further investigate the underlying molecular mechanisms of PAXIP1-AS1 in PAH, the relationship between PAXIP1-AS1 and ETS1/ WIPF1 was studied. Firstly, the expression of WIPF1 was determined in both in vivo and in vitro PAH models and the results showed that the expression of WIPF1 was remarkably elevated compared with the control in both in vivo and in vitro PAH models ( Figure 3A,B). Pearson correlation analysis also revealed that expression of WIPF1 was positively correlated with PAXIP1-AS1 in MCT-induced rat lung tissues ( Figure 3C). Moreover, the data of Western blot analysis determined the positive regulation of PAXIP1-AS1 on WIPF1 expression. As expected, PAXIP1-AS1 overexpression significantly enhanced the expression of WIPF1, while the inhibition of PAXIP1-AS1 led to the opposite result ( Figure 3D).
The data of gene co-expression network using Coexpedia database showed that WIPF1 ranked in the first place of the potential genes co-expressed with ETS1. 26 We further found that ETS1 has a binding region in the promoter of WIPF1 with JARSPAR database.
To further confirm the regulatory effects of PAXIP1-AS1 on WIPF1, RIP assay was conducted and the data verified the direct binding relationship between PAXIP1-AS1 and ETS1 ( Figure 3E). Besides, dual-luciferase reporter assay also showed that the luciferase activity was remarkably higher when ETS1 was overexpressed in WT-WIPF1; however, no significant difference was found in MUT-WIPF1 ( Figure 3F,G). Additionally, ChIP analysis demonstrated that ETS1 directly bound to the promoter region of WIPF1 and the enrichment of ETS1 markedly decreased when PAXIP1-AS1 was suppressed ( Figure 3H,I). Further, Western blotting assay further validated that overexpression of PAXIP1-AS1 led to significant up-regulation of ETS1 and WIPF1, while these effects were abolished by silencing ETS1 (Figure 3J). All these findings suggested that PAXIP1-AS1 positively regulated the expression of WIPF1 through assembling ETS1 protein.

| PAXIP1-AS1 promoted cell viability and migration of hPASMCs through ETS1/WIPF1 signalling
Then, PAXIP1-AS1, ETS1 and WIPF1 were co-overexpressed or suppressed and cell viability and migration were measured. As shown in Figure 4A, it was found that the hypoxia condition remarkably enhanced cell viability of hPASMCs. Overexpression of PAXIP1-AS1 markedly promoted cell viability, while either inhibition of ETS1 or WIPF1 reversed the effects under both in hypoxia and in normal conditions. The data of BrdU assay and immunofluorescence of Ki67 exerted the similar results (Figure 4B-E).
Consistently, wound healing assay also presented that overexpression of PAXIP1-AS1 apparently facilitated the cell migrative ability, while either inhibition of ETS1 or WIPF1 reversed the positive effects ( Figure 4F), which was also confirmed by transwell assay

| WIPF1 interacted and regulated RhoA
To further investigate the mechanism of WIPF1 in PAH, the relationship between WIPF1 and RhoA was determined. Results found that the expression of WIPF1 and RhoA was positively correlated in PAH rat model by Pearson's analysis ( Figure 5A). When WIPF1 was knocked down, the expression of WIPF1 was remarkably downregulated; however, no significant difference was found in mRNA levels of RhoA ( Figure 5B). Interestingly, WIPF1 suppression induced remarkable down-regulation of the protein levels of RhoA ( Figure 5C,D), indicating that WIPF1 regulated the post-translational level of RhoA. It was also found that the decrease of RhoA by suppression of WIPF1 was rescued by proteasome inhibitor MG132 ( Figure 5E), indicating that RhoA undergoes proteasomal degradation in the absence of WIPF1. Additionally, Co-IP analysis demonstrated that WIPF1 could interact with RhoA ( Figure 5F). up-regulated in PAH model; however, sh-PAXIP1-AS1 remarkably down-regulated its expression, which was also decreased by fasudil ( Figure 6D). Western blot analyses also implied that the PAHinduced high expression of WIPF1, ETS1 and RhoA was inhibited by suppression of PAXIP1-AS1, and inhibition of RhoA by fasudil also led to inhibition of the expression of WIPF and ETS1 ( Figure 6E).

| D ISCUSS I ON
PAH, which has a reported incidence of 1.1-17.6 per million adults each year, is a rare disorder with high mortality rate. 29 The aetiol-  To further investigate the molecular mechanism of PAXIP1-AS1 in PAH, the relationship between PAXIP1-AS1 and ETS1 was confirmed, and we also demonstrated that the levels of ETS1 were increased in PAH rats. Actually, the relationship between ETS1 and PAXIP1-AS1 has been previously observed in the above study in glioma. 22 Besides, ETS1 was also found to promote proinflammatory responses and neointima formation in carotid artery endoluminal vascular injury. 31 In lung diseases, ETS1 acts as an oncogene and could promote cell viability and migration of lung cancer cells. 26 However, no study reported the role of ETS1 in the pathogenesis of PAH. In this study, we investigated that ETS1 was up-regulated in PAH and could also promote cell viability and migration of hypoxia- were increased, leading to the down-regulated of p27, which contributed to the PASMC proliferation. 35 In the present research, we also found that RhoA was activated in PAH models and we firstly found RhoA could be activated by WIPF1. Fasudil, a RhoA-ROCK inhibitor, is commonly used as a vasodilator in cerebral vasospasm, which is also seen as a therapeutic agent in PAH treatment. 36 Fasudil is reported to inhibit RhoA signalling in many diseases such as cardiomyopathy and osteosarcoma. 37,38 A single dose of Rho kinase inhibitor fasudil was reported to remarkedly reduce the mean pulmonary arterial pressure and vascular resistance in a sample of nine patients with severe PAH. 39 Similarly, our study also confirmed that RhoA was obviously up-regulated in lung tissues with PAH, contributing to PASMC proliferation, while the application of fasudil or inhibition of PAXIP1-AS1 attenuated these effects. These results are consistent with the above studies. Besides, we also found RhoA was a downstream target of WIPF1, which was also observed in a previous research in cancer. 40 The present study also has some limitations. Firstly, deeply molecular mechanism of how PAXIP1 was regulated is still unclear.
Secondly, clinical significance of PAXIP1-AS1 should be further confirmed in PAH patients. All these need more studies to illustrate.
To conclude, our study revealed a novel mechanism by which PAXIP1-AS1/ETS1/WIPF1/RhoA axis regulated the development of PAH, helping better understanding the pathology of PAH and further validating PAXIP1-AS1 as a therapeutic target for PAH treatment.

ACK N OWLED G EM ENTS
We would like to show our sincere gratitude to the reviewers for their constructive comments. This work is supported by National Clinical Key Specialist Construction Project.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article. The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.

E TH I C A L A PPROVA L
All animal care and experimental procedures were approved by the Animal Care Ethics and Use Committee of the Second Xiangya Hospital of Central South University and performed in accordance with the guidelines of this Committee.

CO N S E NT FO R PU B LI C ATI O N
Not applicable. This article does not contain any studies with human participants performed by any of the authors.